Click-to-chelate : Design and incorporation of triazole-containing metal-chelating systems into biomolecules of diagnostic and therapeutic interest

Article abstract of DOI:10.1002/chem.200702024

The site-specific conjugation of metal chelating systems to biologically relevant molecules is an important contemporary topic in bioinorganic and bioorganometallic chemistry. In this work, we have used the Cu'-catalyzed cycloaddition of azides and terminal alkynes to synthesise novel ligand systems, in which the 1,2,3-triazole is an integral part of the metal chelating system. A diverse set of bidentate alkyne building blocks with different aliphatic and aromatic backbones and various donor groups were prepared. The bidentate alkynes were reacted with benzyl azide in the presence of a catalytic amount of Cu ¹ to form tridentate model ligands. The chelators were reacted with [ReBr₃(CO)₃]^2- to form well-defined and stable complexes with different overall charges, structures and hydrophilicities. In all cases tridentate coordination of the ligands, including through N3 of the 1,2,3-triazole ring, was observed. The ligand systems could also be quantitatively radiolabelled with the precursor [^99mTc (H ₂O)₃(CO)₃]⁺ at low ligand concentrations. Similarly the alkynes were reacted with an azido thymidine derivative to form a series of compounds, which could be radiolabeled in situ to form single products. Subsequent incubation of the neutral and cationic organometallic ^99mTc thymidine derivatives with human cytosolic thymidine kinase, a key enzyme in tumour proliferation, revealed that only the neutral compounds maintained substrate activity towards the enzyme. Bioconjugation, radiolabelling and enzymatic reactions were successfully performed in a matter of hours. Thus, click chemistry provides an elegant method for rapidly functionalising a biologically relevant molecule with a variety of efficient metal chelators suitable for (radiolabelling with the M(CO) ₃ core (M = ^99mTc, Re), to offer new potential for technetium-99m in clinical and preclinical tracer development.

Full text of DOI:10.1002/chem.200702024

FULL PAPER
DOI: 10.1002/chem.200702024
“Click-to-Chelate”: Design and Incorporation ofTriazole-Containing Metal-
Chelating Systems into Biomolecules ofDiagnostic and Therapeutic Interest
Harriet Struthers,[a, b] Bernhard Spingler,[c] Thomas L. Mindt,[b] and Roger Schibli*[a, b]
Abstract: The site-specific conjugation
of metal chelating systems to biologi-
cally relevant molecules is an impor-
tant contemporary topic in bioinor-
ganic and bioorganometallic chemistry.
In this work, we have used the CuI-cat-
alyzed cycloaddition of azides and ter-
minal alkynes to synthesise novel
ligand systems, in which the 1,2,3-tria-
zole is an integral part of the metal
chelating system. A diverse set of bi-
dentate alkyne building blocks with dif-
ferent aliphatic and aromatic back-
bones and various donor groups were
prepared. The bidentate alkynes were
reacted with benzyl azide in the pres-
ence of a catalytic amount of CuI to
form tridentate model ligands. The che-
[ReBr3(CO)3]2À to form well-defined
and stable complexes with different
overall charges, structures and hydro-
philicities. In all cases tridentate coor-
dination of the ligands, including
through N3 of the 1,2,3-triazole ring,
was observed. The ligand systems
could also be quantitatively radiola-
form single products. Subsequent incu-
bation of the neutral and cationic or-
ganometallic 99mTc thymidine deriva-
tives with human cytosolic thymidine
kinase, a key enzyme in tumour prolif-
eration, revealed that only the neutral
compounds maintained substrate activi-
ty towards the enzyme. Bioconjugation,
radiolabelling and enzymatic reactions
belled with the precursor
[
99mTc
G
were successfully performed in
a
trations. Similarly the alkynes were re-
acted with an azido thymidine deriva-
tive to form a series of compounds,
which could be radiolabelled in situ to
matter of hours. Thus, click chemistry
provides an elegant method for rapidly
functionalising a biologically relevant
molecule with a variety of efficient
metal chelators suitable for (radio)lab-
elling with the M(CO)3 core (M=
99mTc, Re), to offer new potential for
technetium-99m in clinical and preclin-
ical tracer development.
Keywords: click chemistry · ligand
design · rhenium · technetium ·
thymidine
lators
were
reacted
with
Introduction
mography (SPECT). In recent years, the focus has mainly
been on positron emitting isotopes such as fluorine-18
(T1/2=109.8 min) and carbon-11 (T1/2 =20.5 min) suitable for
PET. Radiolabelling methods for carbon-11 and fluorine-18
are established, but still challenging, typically requiring mul-
tistep syntheses, and are by no means quantitative, despite
semi- or fully-automated synthesisers.[1] Furthermore, as a
result of their short half-lives, such isotopes have to be pro-
duced on site by expensive cyclotrons and require extensive
laboratory infrastructures, which together prohibit a more
widespread application of PET. Radionuclides with suitable
decay characteristics for in vivo SPECT are usually more
readily available and often have longer half-lives (from sev-
eral hours to several days), which facilitate their handling
and processing. In addition, over the last few years, preclini-
cal small animal SPECT scanners have become available
with sub-millimetre spatial resolution and excellent sensitivi-
ty, which outperform comparable PET devices.[2] It is antici-
pated that this will drive an increased interest in novel
SPECT tracers in preclinical research.
The radiolabelling of biologically active molecules has
become an important tool for the non-invasive assessment
of novel drug candidates as a result of the high sensitivity of
nuclear imaging technologies such as positron emission to-
mography (PET) and single photon emission computed to-
[a] H. Struthers, Prof. R. Schibli
Center for Radiopharmaceutical Science ETH-PSI-USZ
Paul Scherrer Institute, 5232 Villigen (Switzerland)
Fax : (+41)56-310-2849
E-mail: roger.schibli@pharma.ethz.ch
[b] H. Struthers, Dr. T. L. Mindt, Prof. R. Schibli
Department of Chemistry and Applied Biosciences
ETH Zurich, 8093 Zurich (Switzerland)
[c] Dr. B. Spingler
Institute of Inorganic Chemistry
University of Zurich, 8057 Zurich (Switzerland)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6173

One of the most prominent single photon emitting radio-
nuclides is technetium-99m (T1/2 =6 h, 140 keV g-radiation),
the mainstay of diagnostic nuclear medicine. Unlike most
PET nuclides, technetium-99m is readily available at low
cost from a 99Mo/99mTc generator system. However, whereas
non-metal radionuclides can be covalently attached to the
target molecule, stable incorporation of a radiometal such as
technetium into a biomolecule requires an appropriate bi-
functional chelating agent and an efficient strategy to assem-
ble the desired bioconjugate. Numerous functionalisation
strategies and bifunctional chelating systems have been re-
ported.[3,4] However, syntheses are invariably multistep, fre-
quently inefficient, and often require protecting group
chemistry to prevent unwanted side-reactions during incor-
poration into the targeting molecule. These are all critical
issues, which have to be properly addressed if novel metal
based SPECT tracers are to become more widespread, par-
ticularly in preclinical research.
Results and Discussion
Ligand Design and Synthesis: The technetium/rhenium tri-
carbonyl core forms highly stable, low spin d6 complexes
with a wide range of ligand systems.[14–17] We have shown
that coordination of tridentate chelating systems to the
M(CO)3 core efficiently protects the metal centre from
ligand-exchange reactions, for example, with functional
groups of plasma proteins, an observation that leads to
better pharmacokinetic profiles for 99mTc(CO)3 complexes
with tridentate ligands than for complexes with bidentate li-
gands.[18] The incorporation of different tridentate chelating
systems into azido-functionalised biomolecules requires the
synthesis of suitable alkyne building blocks. For our intend-
ed purpose and as proof of concept, five suitable alkynes
were synthesised (2, 4–7), and two commercially available
compounds (1, 3) were also investigated (Table 1). The pri-
The use of the CuI-catalyzed cycloaddition of azides and
terminal alkynes (“click chemistry”)[5,6] has only very recent-
ly found applications in the design of ligands for transition
metals.[7–13] This is surprising given the attractiveness of click
chemistry: The CuI-catalyzed cycloaddition gives high chem-
ical yields under mild reaction conditions even in aqueous
media; the reactions are regiospecific (resulting exclusively
in the formation of 1,4-bifunctionalised 1,2,3-triazole prod-
ucts); finally an additional, but often neglected, feature of
the clicked products is that the 1,2,3-triazole itself is an ef-
fective ligand for various transition metals. Our group has
recently reported the synthesis of histidine-like chelating
systems in which the imidazole ring has been replaced by a
1,4-bifunctionalised 1,2,3-triazole through the reaction of an
azide- or an azide-containing biomolecule with propargyl
glycine.[13] Their organometallic 99mTc(CO)3 complexes
proved to be stable in vivo. An appealing aspect of this ap-
proach, particularly for potential biomedical applications, is
that the click reaction allows simultaneous formation of the
chelating system and conjugation to a biomolecule in a
single high-yielding step.
Table 1. EC50 values for ligands L1–L7.
Alkyne
Ligand
EC50[a] [m]
1
2
L1
L2
2.3”10À7
2.1”10 À7
3
L3
>1”10À3
4
5
6
L4
L5
L6
5.8”10À8
n/a[b]
3.5”10À7
7
L7
n/a[c]
[a] Ligand concentration necessary to achieve 50% radiolabelling yield if
reacted with [99mTc(CO)3(H2O)3]+. [b] Not obtained owing to high lipo-
We have now extended the scope of the “click-to-chelate”
approach. In this work we show that by combining both
newly synthesised and commercial, clickable azide and
alkyne building blocks with different substituents, various
1,2,3-triazole containing polydentate ligands can be effi-
ciently synthesised. The novel chelating systems give rise to
metal complexes of different size, overall charge and hydro-
philicity when (radio)labelled with the M(CO)3 core (M=
99mTc, Re). For proof of concept we applied the modular ap-
proach to the parallel synthesis and radiolabelling of a series
of organometallic thymidine derivatives. The resulting metal
complexes were tested for substrate activity towards human
cytosolic thymidine kinase (hTK1). Using this strategy we
were able to identify novel organometallic substrates for
hTK1and qualitatively analyse structure-activity relation-
ships in a matter of hours starting from the organic azide/
AHCTREUNG
philicity of ligand and/or complex. [c] Two products are formed.
mary considerations for the synthesis of suitable alkynes
were first structural and steric variation of the ligand (and
subsequent complexes), as well as the potential to form
complexes with different overall charges and varied hydro-
philicity.
Na-Propargyl glycine 2, was prepared by alkylation of
propargyl amine with methyl bromoacetate followed by
ester hydrolysis with sodium hydroxide (1m). 2-Propargyl
malonate dimethyl ester is commercially available; the
esters were hydrolysed with four equivalents of sodium hy-
droxide (1m) to give 3. Alkynes 4 and 5 were prepared in a
single step by reductive alkylation of propargyl amine with
2-pyridine carboxaldehyde or 2-thiophene carboxaldehyde,
respectively. Selective S-alkylation of 2-(amino)ethanethiol
alkyne building blocks and commercial Na[
99mTcO4].
A
6174
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6173 – 6183

Incorporation of Metal Chelating Systems into Biomolecules
FULL PAPER
and cysteine with propargyl bromide was achieved using
Boc protected precursors; the Boc protecting groups were
removed using a 9:1mixture of dichloromethane and tri-
fluoroacetic acid to give 6 and 7, respectively. (See Experi-
mental Section for further details.)
Compounds 1–6 provide structural variation and a range
of donor atoms. Compound 7 is a bifunctional building
block, which in addition to being coupled to an azide, can
also be coupled to a second molecule of biological interest
through either the amine or carboxylic acid group, while still
providing a tridentate metal chelating system.
carboxylic acid groups were protected as methyl esters and
in the synthesis of L1, L6 and L7, primary amines were Boc
protected to simplify purification. The yields of the click re-
action were typically 60–90% after purification. In all cases
the syntheses could also be carried out without protecting
the carboxylate and amine groups, but this prohibited effi-
cient purification of the product.
Re(CO)3 complex formation: Rhenium tricarbonyl com-
plexes of all ligands were readily prepared as outlined in
Scheme 2 by heating one equivalent of [ReBr3(CO)3][NEt4]2
A
Each of the alkyne building blocks was reacted with
benzyl azide to form the model triazole containing ligands
L1–L7 (Scheme 1). The [3+2] cycloaddition reactions were
with one equivalent of the ligand in methanol (L4, L5, L7)
or a mixture of methanol and water (L1, L2, L3, L6). In all
cases, HPLC analyses of the crude reaction mixtures re-
vealed quantitative formation of a single product after 2 h at
508C. Complexes [Re(CO)3L1], [Re(CO)3L3]NEt4, [Re
(CO)3L4]Br, [Re(CO)3L6]Br and [Re(CO)3L7]Br were puri-
fied using reverse phase Sep-Pak columns, whereas analyti-
cally pure samples of [Re(CO)3L2] and [Re(CO)3L5]Br pre-
cipitated from their reaction mixtures directly. Although
HPLC analysis confirmed quantitative formation of the
products, the isolated yields varied between 40% and 80%,
depending on the purification method. All of the complexes
were characterised by using mass spectrometry, IR spectros-
copy, NMR spectroscopy and elemental analysis. Crystals
suitable for X-ray structure determination could be obtained
for complexes [Re(CO)3L2], [Re(CO)3L3]NEt4 and [Re
(CO)3L6]Br.
Scheme 1. General synthesis of ligands L1–L7 via the CuI-catalysed [3+
2] cycloaddition reaction: a) 0.1equiv. Cu (OAc)2, 0.1equiv. sodium ascor-
A
bate, tBuOH/H2O, 12 h, room temperature. For R see Table 1.
performed using similar conditions to those reported by the
Sharpless group.[6] One equivalent of azide and one equiva-
lent of alkyne were stirred at room temperature for 12 h in
a mixture of tertiary butanol and water with 0.1equivalents
of copper acetate and 0.2 equivalents of sodium ascorbate
to generate CuI in situ. For the synthesis of L2 and L3 the
Scheme 2. Synthesis and structures of Re(CO)3 complexes.
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6175

R. Schibli et al.
Comparison of the NMR spectra of the complexes with
those of the free ligands revealed characteristic differences
in chemical shifts and coupling patterns, which allowed as-
signment of the chemical composition and structure of the
chelates. Signals in the proton NMR spectra of the com-
plexes typically occur at higher frequencies than the corre-
sponding signals in the free ligand as a result of coordina-
tion to the electron-deficient metal centre. In [Re(CO)3L1],
the bCH2 protons are no longer magnetically equivalent,
and independent resonances are observed for these protons
as well as a more complex coupling pattern for the aCH
proton, suggesting facial coordination of the tridentate
ligand. Similarly, in [Re(CO)3L2] and [Re(CO)3L4–L7]Br,
four distinguishable resonances result from the two CH2
groups of the chelate rings. In addition the structures of
[Re(CO)3L4]Br and [Re(CO)3L7]Br are sufficiently rigid in
solution that two independent signals are observed for the
benzylic CH2 protons.
In the proton NMR spectra of [Re(CO)3L1] and
[Re(CO)3L4–L7]Br recorded in [D4]MeOH, protons of the
coordinated amine are also observed, as coordination of the
amine to the metal results in much slower H/D exchange
rates than in the free ligand. Similar behaviour has been re-
ported for 99Tc/Re(CO)3 complexes containing other pri-
mary and secondary amines.[18,19] Differences between the
proton NMR spectra of the free ligand L3 and [Re
(CO)3L3]NEt4 are less pronounced than for all other com-
plexes, as a result of the higher symmetry of this complex
(Cs compared to C1 in all other complexes). The different
possible diastereoisomers of the Re(CO)3-complexes with
the ligands L2, L4, L5, L7 could not be distinguished in the
proton NMR spectra.
The reaction of the cysteine based ligand L7 gave the
complex, [Re(CO)3L7]Br, with kN3,kS,kN coordination of
the ligand and the methyl ester still intact, as evident from
NMR analyses. This type of coordination models the situa-
tion in which L7 is coupled through the carboxylic acid to
another molecule of interest. The hydrolysed ligand L7b
(L7, but without the ester group) can potentially form three,
structurally different complexes with the M(CO)3 core: two
in which the ligand is coordinated through the N3 of the tri-
azole (see Scheme 1for numbering scheme), sulfur and
either the primary amine (kN3,kS,kN coordination) or the
carboxylate (kN3,kS,kO coordination), and one in which the
ligand is coordinated through the sulfur, primary amine and
carboxylate (kS,kN,kO coordination). However, the reaction
Scheme 3. Reaction of L7b with [ReBr3(CO)3][NEt4]2.
A
The complex with kS,kN,kO coordination (40%) was simi-
larly identified by coordination of the primary amine but
the equivalence in this case of the benzyl CH2 protons. The
formation of two products is interesting given that with a
structurally similar ligand, 2(-2’pyridyl)ethyl-cysteine, Kara-
giorgou et al. observed exclusive formation of a single com-
plex with kS,kN,kO coordination, without coordinative par-
ticipation of the pyridyl nitrogen.[20]
The X-ray structure analyses of [Re(CO)3L2], [Re
(CO)3L3]NEt4 and [Re(CO)3L6]Br (Figure 1) confirmed the
tridentate, facial coordination of the ligands to the metal
centre as expected from NMR analyses. All three structures
show that the 1,2,3-triazole ligand is coordinated through
N3. This is consistent with DFT calculations we have report-
ed previously, which predict that the highest electron density
is at position N3 (favouring N3 coordination) followed by
N1, with the lowest at position N2 of the triazole ring.[13]
The bond lengths between rhenium and N3 of the triazole
are between 2.15 and 2.21 Š, which is in good agreement
À
with the Re N bond length in the bi-1,2,3-triazole contain-
ing complex [ReCl
A
bi-1H-1,2,3-triazole) (2.176(6) Š).[12] The bond lengths are
À
also comparable with the Re N bond lengths in similar
Re(CO)3 complexes with imidazole or pyrazole based li-
gands, which are typically 2.19–2.21 Š.[16,21] Crystallographic
data for all structures solved are reported in Table 2.
Radiolabelling ofL1 –L7 with [99 mTc(H2O)3(CO)3]+: Partic-
E
ularly for receptor targeting radiopharmaceuticals, it is im-
portant that high labelling yields can be achieved at low
ligand concentration to avoid receptor saturation. Thus, po-
tential ligand systems should form defined and stable tech-
netium complexes at micromolar concentrations or lower.
The technetium-99m complexes of ligands L1, L2, L4 and
of L7b with [ReBr3(CO)3][NEt4]2 led to the formation of
A
only two products after heating in methanol for 2 h as
shown by HPLC analysis (Scheme 3). Elemental analysis of
the purified mixture of products revealed both complexes
were neutral. The structures of the products were elucidated
from the proton NMR spectrum. The compound with
kN3,kS,kN coordination (60%) was identified by signals for
the amine protons as observed for [Re(CO)3L1] and
[Re(CO)3L4–L7]Br and two independent resonances for the
benzylic CH2 protons, attributed to coordination of the tria-
zole, as observed for [Re(CO)3L4]Br and [Re(CO)3L7]Br.
L6 were prepared by adding
a solution of [
99mTc-
AHCTREUNG
phosphate buffer (PBS) at pH 7.4, and characterised by
comparison of their g-HPLC traces with the UV-HPLC
traces of the corresponding rhenium complexes. Labelling
yields were assessed as the ligand concentration was varied
between 10À3 and 10À8 m, to give rise to step-sigmoid curves
6176
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6173 – 6183

Incorporation of Metal Chelating Systems into Biomolecules
FULL PAPER
AHCTREUNG
uct. The nature of the reaction with [99mTc(H2O)3(CO)3]+
A
was surprising given that a well-defined and characterisable
Re(CO)3 complex could be synthesised, and we do not cur-
rently have a plausible explanation for the unusual behav-
iour. The problems we encountered with L3 unfortunately
precluded the use of building block 3 in our further investi-
gations. L5 is noticeably more lipophilic than all of the
other triazole containing ligands, which complicated label-
ling with the 99mTc(CO)3 core in aqueous media and analysis
of the technetium-99m complex formed by reverse phase
HPLC. The radiolabelling of L7 also led to the formation of
two products, neither of which corresponded to the rhenium
complex [Re(CO)3L7]. The g-HPLC trace in fact matched
the UV-HPLC trace of the reaction between ligand L7b and
the rhenium precursor as shown in Scheme 3, which suggest-
ed that ligand L7 was hydrolysed under the conditions used
for technetium-99m labelling. Radiolabelling of L7b con-
firmed this assumption.
Rapid identification of substrates for hTK1 using in situ
clicked and radiolabelled thymidine derivatives: Click
chemistry has already proved an effective tool for the paral-
lel synthesis of large numbers of compounds for biological
assays, and allows structure-activity information to be ob-
tained without multistep syntheses or large amounts of ma-
terial.[23] These are features that are also attractive in radio-
pharmaceutical development. In the context of the following
experiments, it is noteworthy that the reaction between
[
99mTc(H2O)3(CO)3]+ and the potentially bidentate alkyne
A
building blocks 1–6 did not result in the formation of well-
defined or stable complexes at a comparably low concentra-
tion to the tridentate, triazole ligands. The same holds true
for the azide component of the click reaction. Thus, the
presence of the triazole as a donor group appears to greatly
increase the structural uniformity and stability of the result-
ing 99mTc(CO)3 complexes. As a consequence, there is a
unique opportunity to expedite tracer development, because
even in presence of excess alkyne or azide, the click product
is preferentially labelled. We have previously reported a
one-pot, two-step procedure in which the crude click reac-
Figure 1. ORTEP-3[22] representations of the neutral complex
A) [Re(CO)3L2], the complex anion B) [Re(CO)3L3]À and the complex
cation C) [Re(CO)3L6]+, with thermal ellipsoids shown at 50% probabil-
ity. Hydrogen atoms are omitted for clarity. Selected bond lengths [Š]
À
À
and angles [8]: [Re(CO)3L2] Re(1) C(1) 1.919(3), Re(1) C(2) 1.912(2),
À
À
À
Re(1) C(3) 1.897(3), Re(1) N(3) 2.1511(18), Re(1) N(4) 2.2290(17),
À
À
À
À
À
Re(1) O(5) 2.1405(16), C(1) Re(1) N(3) 171.96(8), C(3) Re(1) N(3)
À
À
À
À
À
92.75(9), C(2) Re(1) N(4) 170.28(9), N(3) Re(1) N(4) 76.53(7), C(3)
À
À
À
À
À
Re(1) O(5) 174.60(8), C(2) Re(1) O(5) 94.17(8), N(3) Re(1) O(5)
À
À
82.46(7); [Re(CO)3L3]NEt4 Re(1) C(1) 1.905(3), Re(1) C(2) 1.906(3),
À
À
À
Re(1) C(3) 1.898(3), Re(1) O(5) 2.118(2), Re(1) O(7) 2.1383(18),
À
À
À
À
À
Re(1) N(3) 2.199(2), C(1) Re(1) N(3) 176.12(11), C(3) Re(1) N(3)
À
À
À
À
94.34(10), C(2) Re(1) O(5) 175.96(10), N(3) Re(1) O(5) 84.87(8),
tion mixtures are labelled directly with the
(H2O)3(CO)3]+ precursor.[13] The resulting products and ra-
[
99mTc
À
À
À
À
À
À
C(3) Re(1) O(7) 174.75(10), C(2) Re(1) O(7) 96.29(9), N(3) Re(1)
À
À
ACHTREUNG
O(7) 83.06(8); [Re(CO)3L6]Br Re(1) C(1) 1.909(2), Re(1) C(2)
À
À
À
1.932(2), Re(1) C(3) 1.936(2), Re(1) N(3) 2.1799(16), Re(1) N(4)
2.2074(17), Re(1) S(1) 2.4753(5), C(1) Re(1) N(3) 172.28(8), C(3)
diolabelling yields were found to be almost identical to
those when the reactions were performed with the pre-puri-
fied, triazole containing ligands, but the one-pot procedure
avoids unnecessary purification steps. Thus, with in situ radi-
olabelling, click chemistry can rapidly provide a set of com-
pounds for preliminary in vitro screening and assessment.
As proof of the value of this one-pot procedure, we aimed
to synthesise, radiolabel and assess in vitro a set of novel
99mTc(CO)3-thymidine complexes, in order to identify the
structural and physico-chemical parameters necessary to
maintain activity towards hTK1. Technetium labelled thymi-
dine analogues have the potential to act as substrates for
À
À
À
À
À
À
À
À
À
Re(1) N(3) 93.59(8), C(2) Re(1) N(4) 177.83(7), N(3) Re(1) N(4)
À
À
À
À
À
84.16(6), C(3) Re(1) S(1) 170.94(6), C(2) Re(1) S(1) 97.17(6), N(3)
À
Re(1) S(1) 78.23(4).
(see the Supporting Information). This allowed the determi-
nation of EC50 values (ligand concentration necessary to
achieve 50% labelling yield; Table 1). Ligands L1, L2, L4
and L6 proved very efficient, with EC50 values in the sub-
micromolar range, which are comparable to values reported
for Nt-functionalised histidine derivatives.[13,16] The radio-
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6177

R. Schibli et al.
human cytosolic thymidine kinase (hTK1) and therefore as
markers for cancer cell proliferation, since hTK1shows a
higher than normal expression in a wide variety of cancer
cells.[24] However, for unknown reasons no Tc/Re-labelled
thymidine derivates tested to date have shown substrate ac-
tivity.[25] It is known that modification of thymidine at posi-
tion N3 of the pyrimidine base does not necessarily affect its
ability to act as a substrate for hTK1. Therefore we synthe-
[ReBr3(CO)3]2À, which was carried out under the same con-
ditions (analysis by MS).
The ability of the thymidine analogues to act as substrates
for hTK1, and the influence of overall charge and/or struc-
ture of the complexes on phosphorylation were investigated
with the technetium labelled compounds as shown schemati-
cally in Figure 2. The complexes were purified by HPLC
sised N3 functionalised azido thymidine (dT-N3)
(Scheme 4). The synthesis is straightforward and can be
Scheme 4. Functionalization of thymidine at the N3 position:
a) TBDMSCl, imidazole, DMF, 98% yield, b) BrCH2CH2Br, Cs2CO3,
DMF, 95% yield, c) NaN3, MeCN, 80% yield, d) NBu4F, THF, 96%
yield.
readily accomplished in four steps from commercially avail-
able thymidine. The 3’ and 5’ hydroxyl groups were protect-
ed with tert-butyldimethylsilyl groups,[26] to allow selective
alkylation at the N3 position with 1,2-dibromoethane and to
simplify purification. The azido compound is formed via nu-
cleophilic substitution of the bromide with excess sodium
azide, followed by removal of the silyl protecting groups
with tetra
Based on the labelling profiles of the model ligands with
99mTc(H2O)3(CO)3]+, four alkynes (1, 2, 4, 6) were selected
butylammonium fluoride.
Figure 2. One pot click reaction and in situ radiolabelling of thymidine
analogues: a) 0.1equiv Cu (OAc)2, 0.2 equiv sodium ascorbate, 1h, 65 8C;
A
b) [99mTc(OH2)3(CO)3]+, PBS, 1h, 100 8C. g-HPLC traces after incuba-
G
[
ACHTREUNG
tion of A) [99mTc(CO)3dT2] and B) [99mTc(CO)3dT4]+ with ATP in the
for reaction with the azido-thymidine derivative. Stock solu-
tions of each of the four alkynes were prepared in water (1,
2) or methanol (4, 6), along with aqueous solutions (0.01m)
of dT-N3, copper(II) acetate and sodium ascorbate. The
click reaction was performed on a 100 mL scale with one
equivalent of the relevant alkyne, 1.2 equivalents of dT-N3
(0.2 mg), 0.2 equivalents of sodium ascorbate and 0.1equiv-
alents of copper(II) acetate. The reaction mixtures were
heated at 658C for 60 minutes, after which time product for-
mation was confirmed by mass spectroscopy and HPLC.
presence of hTK1.
and added to a stock solution of ATP, MgCl2 and Tris. Re-
combinant hTK1was added, and the solutions were incubat-
ed at 378C for three hours before being analyzed by HPLC.
The g-HPLC traces of the enzymatic reactions of both
neutral complexes, [99mTc(CO)3dT1] and [99mTc(CO)3dT2],
revealed two distinguishable 99mTc containing complexes
after incubation with hTK1(Figure 2A). The first complexes
eluted were identified by co-injection of the starting materi-
The precursor [99mTc(H2O)3(CO)3]+ in PBS buffer (pH 7.4)
A
was added to the crude click solutions. The reaction mix-
tures were heated again at 1008C for 60 minutes before
product formation was confirmed by g-HPLC. Labelling of
dT1, dT2, dT4 and dT6 was extremely efficient, with yields
greater than 90% in all cases. The identity of the products
was inferred from the successful labelling of the ligands with
al
[
as
the
phosphorylated
thymidine
analogues
99mTc(CO)3dT1MP] and [99mTc(CO)3dT2MP]. Phosphory-
lation of the corresponding Re complex [Re(CO)3dT2] to
[Re(CO)3dT2MP] was confirmed by MS analysis. For both
[
99mTc(CO)3dT1] and [99mTc(CO)3dT2] 20Æ5% of the start-
ing material was converted into the mono-phosphate prod-
6178
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6173 – 6183

Incorporation of Metal Chelating Systems into Biomolecules
FULL PAPER
Sep-Pakꢁ columns (Waters) were washed with methanol and water prior
to use.
uct. In contrast neither of the positively charged complexes
showed any phosphorylation by hTK1under the same con-
ditions (Figure 2B). This suggests that in the case of our or-
ganometallic 99mTc/Re thymidine complexes, overall charge
rather than exact structure is more important in determining
their ability to act as substrates for hTK1. In vitro and in
silico experiments are ongoing in order to quantitatively
assess this phenomenon.
Infrared spectra were recorded on a Perkin–Elmer Spectrum BX II FT-
IR, with a Pike MIRacle(TM) ATR accessory. Nuclear magnetic reso-
nance spectra were recorded with a 300 MHz Varian Gemini 2000 spec-
trometer with solvent signals as an internal standard. Chemical shifts (d)
are reported in parts per million (ppm) relative to tetramethylsilane
(0.00 ppm). Values of the coupling constant, J, are given in Hertz (Hz).
The following abbreviations are used for the description of 1H NMR
spectra: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
doublet of doublets (dd), broad singlet (bs). The chemical shifts of com-
plex multiplets are given as the range of their occurrence. Low resolution
mass spectra (MS) were recorded with an LCT Premier ESI-TOF instru-
ment from Waters, using either the negative or positive ionization mode.
High resolution mass spectra (HR-MS) were recorded with a Bruker
FTMS 4.7T BioAPEXII (ESI).
Conclusion
In this work we present an elegant strategy for the synthesis
of 1,2,3-triazole containing ligands in which the 1,2,3-triazole
is an integral part of the metal chelating system, while si-
multaneously coupling them site-specifically to azide con-
taining organic molecules. The “click-to-chelate” approach
represents a significant improvement to classical methods
for the incorporation of metal chelating systems into bio-
molecules, as the reactions are almost quantitative and do
not require protecting group chemistry. The potential of this
strategy was successfully demonstrated by the parallel syn-
thesis of a set of thymidine analogues with different chelat-
ing systems, which could be radiolabelled in situ to form
technetium complexes of different size and overall charge.
Subsequent incubation of the radiolabelled thymidine deriv-
atives with human cytosolic thymidine kinase enabled iden-
tification of the first metal containing substrates ever report-
ed for this enzyme. We have shown that the synthesis and
incorporation of different metal chelating systems and sub-
sequent radiolabelling do not have to be the rate determin-
ing steps in the development of radiopharmaceuticals; by
making functionalization of targeting molecules fast, effi-
cient and predictable, click chemistry could play a crucial
role in the future in expediting the development of potential
SPECT tracers.
CAUTION: 99mTc is a g-emitter (140 keV) with a half-life of 6.01 h. All
reactions involving 99mTc were performed in a laboratory approved for
the handling of radioisotopes, and normal safety procedures were fol-
lowed at all times to prevent radioactive contamination.
N-Propargyl-glycine methyl ester (2a): Propargyl amine (1.5 mL,
23 mmol), methyl bromoacetate (3.3 mL, 35 mmol) and triethylamine
(4.9 mL, 35 mmol) were stirred in MeCN (75 mL) at 508C. After 12 h the
solvent was removed, and the residue redissolved in CH2Cl2. The crude
product was purified by column chromatography with CH2Cl2 and
MeOH (4%). The product was isolated as a yellow oil (2.80 g, 94%). IR
n˜ =3286, 1737, 1437, 1374, 1201, 1139, 1000, 910, 769, 654 cmÀ1
;
1H NMR
(CDCl3): d=3.74 (s, 3H), 3.52 (s, 2H), 3.48 (d, 4J
ACHTREUNG
4
2.23 (t, J
A
172.2, 81.1, 72.0, 51.8, 48.9, 37.5 ppm; MS (ES): m/z: 128.06
[C6H9NO2]H+.
N-Propargyl-glycine (2): 2a (105 mg, 0.83 mmol) was dissolved in MeOH
(2 mL) and 2 equivalents of NaOH (1.65 mL, 1m solution) were added.
The mixture was stirred at room temperature for 1hour and followed by
TLC. When all of the starting material had been consumed, the pH was
decreased to approximately 6 with 1m HCl, and the solvent was removed
under vacuum. The product was purified with a Sep-Pak column. The
fractions containing the product were evaporated and the product isolat-
ed as a white powder (61mg, 65%). IR n˜ =3239, 2992, 2780, 2631, 1633,
1614, 1582, 1408, 1308, 931, 904, 741, 705, 669 cmÀ1
;
1H NMR (D2O): d=
(H,H)=
3.99 (d, 4J
G
ACHTREUNG
2.6 Hz, 1H); 13C NMR (D2O): d=172.3, 79.8, 57.3, 49.5 (MeOH refer-
ence), 46.2, 37.7 ppm; MS (ES): m/z: 114.05 [C5H7NO2]H+.
2-Propargyl malonic acid (3): 2-Propargyl malonate dimethyl ester
(200 mg, 1.18 mmol) was dissolved in MeOH (5 mL) and 4 equivalents of
NaOH (4.71mL, 1 m solution) were added. The yellow solution was
stirred at room temperature for 1hour and followed by TLC. When all
of the starting material had been consumed, the pH was decreased to ap-
proximately 6 with 1m HCl to give a colourless solution. The solvent was
removed under vacuum and the product was purified with a Sep-Pak
column. The fractions containing the product were evaporated under
vacuum to give the product as a white powder (126 mg, 75%). IR n˜ =
Experimental Section
General methods: All reagents and solvents were obtained from commer-
cial sources (Sigma–Aldrich, Alfa Aesar, Bachem) and used as supplied
unless stated otherwise. [Re(Br)3(CO)3]
N
[
3274, 1608, 1583, 1439, 1323, 1281, 1175, 62, 851, 668, 628 cmÀ1
(D2O): d=3.32 (t, 3J(H,H)=7.5 Hz, 1H), 2.60 (dd, 3J(H,H)=7.5 Hz, 4J-
(H,H)=2.1Hz, 2H), 2.30 ppm (t, 4J(H,H)=2.1Hz, 1H); 13C NMR
;
1H NMR
generator (Mallinckrodt–Tyco, Petten) with a 0.9% saline solution.
A
ACHTREUNG
Reactions were monitored by means of HPLC or by using thin layer
chromatography (TLC) using precoated silica gel 60 F254 aluminium
sheets, and visualised by using UV absorption or stained with solutions of
ninhydrin or KMnO4. HPLC was performed by using a Merck–Hitachi
L-7000 system equipped with an L-7400 tuneable absorption detector
and a Berthold LB 506 B radiometric detector. Analytical HPLC was
performed by using either an XTerraꢁ column (MSC18, 5 mm, 4.6”
150 mm, Waters) or a Nucleosilꢁ 5 C18 column (5 mm, 4.6”250 mm, Ma-
cherey-Nagel). Two HPLC solvent systems were used. System I used
aqueous triethylammonium phosphate buffer (0.05m), pH 2.25 (solvent
A), methanol (solvent B), and a gradient as follows: 0 to 15 min, 95% A
to 20% A, 1 mL/min; 15 to 20 min 100% A, 1 mL/min. System II used
water with 0.1% trifluoroacetic acid (solvent A), acetonitrile (solvent B)
and a gradient as follows: 0 to 20 min, 97% A to 0% A, 1mL/min; 20 to
22 min, 0% A to 97% A, 1mL/min; 22 to 25 min, 97% A, 1mL/min.
A
ACHTREUNG
(D2O): d=177.6, 83.7, 70.62, 56.6, 49.5 (MeOH reference), 19.9 ppm; MS
(ES): m/z: 143.04 [C6H6O4]H+.
N-Propargyl-pyridine-2-methylamine (4): Propargyl amine (1.29 g,
23.5 mmol) and pyridine-2-carboxaldehyde (0.84 g, 7.9 mmol) were added
to MeOH (40 mL) and stirred at room temperature for 30 min.
NaCNBH3 (0.74 g, 11.8 mmol) was added and the mixture stirred at
room temperature for a further 2 h. The solvent was evaporated, and the
reaction mixture purified by column chromatography with CH2Cl2 and
MeOH (5%). The product was isolated as a dark orange liquid (0.66 g,
57%). IR n˜ =3291, 1661, 1594, 1571, 1476, 1435, 1361, 1246, 1151, 1121,
1
1098, 1050, 1001, 759, 632 cmÀ1; H NMR (CDCl3): d=8.56 (m, 1H), 7.65
(m, 1H), 7.32 (m, 1H), 7.17 (m, 1H), 4.00 (s, 2H), 3.50 (d, 4J
C
4
2.4 Hz, 2H), 2.24 (t, J
A
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6179

R. Schibli et al.
(CDCl3): d=159.3, 149.6, 136.6, 122.6, 122.2, 81.9, 71.8, 53.9, 38.0 ppm;
MS (ES): m/z: 147.08 [C9H10N2]H+.
C
G
N-Propargyl-thiophene-2-methylamine (5): Propargyl amine (2.17 g,
39.5 mmol) and thiophene-2-carboxaldehyde (1.48 g, 13.2 mmol) were
added to MeOH (65 mL) and stirred at room temperature for 30 min.
NaCNBH3 (1.24 g, 26.3 mmol) was added and the mixture stirred at
room temperature for a further 2 h. The solvent was evaporated, and the
reaction mixture purified by column chromatography with CH2Cl2 and
MeOH (2%). The product was isolated as a yellow liquid (0.87 g, 44%).
IR n˜ =3289, 2843, 1607, 1436, 1368, 1331, 1213, 1166, 1097, 1019, 908,
A
852, 828, 697, 652, 636 cmÀ1 1H NMR (CDCl3): d=7.24–7.18 (m, 1H),
;
6.96–6.90 (m, 2H), 4.04 (s, 2H), 3.40 (d, 4J
(H,H)=2.4 Hz, 2H), 1.95 ppm
G
(bs, 1H); 13C NMR (CDCl3): d=142.8, 126.8, 125.5, 124.9, 81.7, 72.0,
46.7, 37.0 ppm; MS (ES): m/z: 152.05 [C8H9NS]H+.
S-Propargyl-2-(Boc-amino)ethanethiol (6a): 2-(Boc-amino)ethanethiol
(1.00 mL, 5.92 mmol) and propargyl bromide (0.45 mL, 5.99mmol) were
added to a solution of triethylamine (1.25 mL, 8.99mmol) in MeCN
(50 mL). The reaction mixture was stirred at 508C and followed by TLC.
After 2 h the solvent was removed and the reaction mixture purified by
column chromatography with a mixture of hexane and EtOAc (20%) to
give the product as a pale yellow liquid (1.01 g, 80%). IR n˜ =3298, 2977,
2930, 2359, 1694, 1508, 1456, 1392, 1366, 1339, 1251, 1163, 1047, 949, 863,
1
781, 734, 635 cmÀ1
(H,H)=6.4 Hz), 3.26 (d, 2H, 4J
6.4 Hz), 2.26 ppm (t, 1H, 4J(H,H)=2.6 Hz); 13C NMR (CDCl3): d=155.9,
;
1H NMR (CDCl3): d=4.90 (bs, 1H), 3.38 (m, 2H, 3J-
A
N
ACHTREUNG
AHCTREUNG
99.3, 80.0, 71.6, 32.2, 28.6, 25.2, 19.2 ppm; MS (ES): m/z: 238.07
[C10H17NO2S]Na+.
S-Propargyl-2-aminoethanethiol (6): 6a (1.12 g, 5.21 mmol) was dissolved
in CH2Cl2/TFA (9:1; 50 mL) and stirred at room temperature overnight.
The solvent was removed and the crude product purified by column chro-
matography with CH2Cl2 and MeOH (9%) to give the TFA salt of the
product as orange oil. The free amine was obtained by dissolving the
TFA salt in 1m sodium hydroxide (10 mL) and extracting into CH2Cl2.
The organic phase was washed twice with water. The aqueous phases
were basified with aqueous NaOH and re-extracted into CH2Cl2. The or-
ganic phases were collected, dried over Na2SO4 and evaporated to give a
pale yellow oil (395 mg, 98%). IR n˜ =3280, 2922, 1657, 1602, 1442, 1414,
;
A
N
1331, 1232, 1184, 1125, 1022, 988, 802, 713, 648 cmÀ1
;
1H NMR (CDCl3):
4
d=3.26 (d, J
N
G
3J
A
N
1.42 ppm (s, 9H); 13C NMR (CDCl3): d=80.2, 71.3, 40.9, 36.0, 19.1 ppm;
MS (ES): m/z: 116.06 [C5H9NS]H+.
S-Propargyl-N-Boc-cysteine methyl ester (7a): N-Boc-cysteine methyl
ester (1.5 mL, 7.29 mmol) was dissolved in DMF (15 mL). Cs2CO3
(2.38 g, 7.30 mmol) and propargyl bromide (0.5 mL, 6.66 mmol) were
added and the mixture was stirred at room temperature and followed by
TLC. After 3 h the reaction mixture was diluted with water (30 mL) and
the product extracted into EtOAc (50 mL). The organic phase was
washed twice with a 1m NaHCO3 solution and dried over Na2SO4. The
crude product was purified by column chromatography with CH2Cl2 and
MeOH (2%) to give a pale yellow solid (1.11 g, 61%). IR n˜ =3373, 3258,
2986, 2955, 1743, 1681, 1518, 1436, 1422, 1395, 1370, 1320, 1296, 1250,
1238, 1206, 1165, 1079, 1042, 1020, 979, 876, 860, 829, 783, 745, 719,
1
690 cmÀ1; H NMR (CDCl3): d=5.35 (d, 1H, 3J
ACHTREUNG
(m, 3J
(H,H)=2.6 Hz, 1H), 3.23 (dd, 2J
3.21–3.04 (m, 3J(H,H)=4.8 Hz, 2J
(H,H)=22.7 Hz, 1H), 2.28 (t, 4J
(H,H)=2.6 Hz, 1H), 1.44 ppm (s, 9H);
A
ACHTREUNG
A
N
ACHTREUNG
A
ACHTREUNG
A
N
13C NMR (CD3OD): d=171.6, 154.6, 80.3, 79.3, 53.2, 52.7, 33.7, 28.4,
19.9 ppm; MS (ES): m/z: 296.07 [C12H19NO4S]Na+.
S-Propargyl-cysteine methyl ester (7): 7a (100 mg, 0.45 mmol) was dis-
solved in CH2Cl2/TFA (9:1; 5 mL) and stirred at room temperature over-
night. The solvent was removed and the crude product purified by
column chromatography with CH2Cl2 and MeOH (5%) to give a yellow
solid (50 mg, 92%). IR n˜ =2961, 1748, 1668, 1524, 1441, 1330, 1245, 1184,
;
1H NMR
1
3
1133, 839, 799, 722, 648 cmÀ1; H NMR (CD3OD): d=4.35 (dd, J
4.6, 8.0 Hz, 1H), 3.87 (s, 3H), 3.41 (d, 4J
(H,H)=2.6 Hz, 2H), 3.36 (dd, J-
(H,H)=14.9 Hz, 3J(H,H)=4.6, 1H), 3.15 (dd, 2J(H,H)=14.9 Hz, 3J-
ACHTREUNG
ACHTREUNG
2
G
U
G
ACHTREUNG
6180
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6173 – 6183

Incorporation of Metal Chelating Systems into Biomolecules
FULL PAPER
(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-malonic acid (L3): L3a (500 mg,
1.65 mmol) was dissolved in a 1:1 mixture of MeOH and water and 4
equivalents of NaOH (264 mg, 6.60 mmol) were added. The mixture was
stirred at room temperature and followed by TLC. After 2 h, the pH was
decreased to approximately 6 with 1m HCl and the solvent removed
under vacuum. The product was purified with a Sep-Pak column. The
fractions containing the product were evaporated and dried under
vacuum to give a white crystalline solid (286 mg, 63%). IR n˜ =2981,
1976, 1731, 1720, 1558, 1431, 1272, 1231, 1174, 1074, 952, 854, 716, 698,
stirred at room temperature overnight. The solvent was removed and the
crude product redissolved in CH2Cl2. The product precipitated as a white
solid, which was washed well with CH2Cl2 and dried under vacuum
(240 mg, 79%). IR n˜ =3081, 2954, 2029, 1743, 1660, 1525, 1455, 1436,
1330, 1268, 1248, 1179, 1135, 1086, 1052, 986, 900, 869, 833, 802, 748, 721,
712, 700, 641 cmÀ1
5H), 5.59 (s, 2H), 4.32 (dd, 3 J
14.8 Hz, 1H), 3.86 (d, 2J(H,H)=14.8 Hz, 1H), 3.81 (s, 3H), 3.17 (dd, 2J-
(H,H)=14.9 Hz, 3J(H,H)=4.5 Hz, 1H), 3.00 ppm (dd, 2J
(H,H)=14.9 Hz,
3J(H,H)=8.1Hz, 1H); 13C NMR (CD3OD): d=169.7, 146.0, 136.7, 130.1,
;
1H NMR (CD3OD): d=7.91(s, 1H), 7.47–7.29 (m,
A
ACHTREUNG
AHCTREUNG
A
N
ACHTREUNG
667, 646 cmÀ1
;
1H NMR (CD3OD): d=7.74 (s, 1H), 7.39–7.25 (m, 4H),
AHCTREUNG
5.55 (s, 1H), 3.73 (t, 1H, 3J
U
G
129.7, 129.2, 124.3, 55.1, 53.9, 53.3, 32.8, 26.7 ppm; HR-MS (ES): m/z:
307.1219 [C14H18N4O2S]H+ (calcd. 307.1223).
S-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-cysteine (L7b): L7 (209 mg,
0.68 mmol) was dissolved in a 1:1 mixture of MeOH and water (5 mL)
and 2 equivalents of NaOH (54 mg, 1.36 mmol) were added. The mixture
was stirred at room temperature for 2 h and followed by TLC. When all
of the starting material had been consumed, the pH was decreased to ap-
proximately 6 with 1m HCl. The white precipitate was collected by filtra-
tion and dried under vacuum (94 mg, 47%). IR n˜ =3060, 1579, 1483,
1413, 1394, 1341, 1302, 1265, 1245, 1212, 1199, 1128, 1104, 1060, 1029,
N-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-pyridine-2-methylamine (L4):
As per general procedure A, with benzyl azide (182 mg, 1.4 mmol) and 4
(200 mg, 1.4 mmol). The crude product was purified by column chroma-
tography with CH2Cl2 and MeOH (10%) to give a brown oil (253 mg,
66%). IR n˜ =2926, 1591, 1570, 1456, 1434, 1329, 1219, 1127, 1049, 1029,
994, 759, 720 cmÀ1 1H NMR (CDCl3): d=8.49 (m, 1H), 7.58 (m, 1H),
;
7.37 (s, 1H), 7.34–7.29 (m, 3H), 7.20–7.23 (m, 3H), 7.10 (m, 1H), 5.45 (s,
2H), 3.89 (s, 4H), 1.79 ppm (bs, 1H); 13C NMR (CDCl3): d=149.3, 136.6,
129.1, 128.7, 128.2, 122.5, 122.1, 121.8, 54.4, 54.1, 44.3 ppm. HR-MS (ES):
m/z: 280.1562 [C16H17N5]H+ (calcd. 280.1557).
1
961, 908, 896, 856, 787, 764, 727, 705, 643, 617 cmÀ1; H NMR (D2O): d=
7.88 (s, 1H), 7.38–7.25 (m, 5H), 5.51 (s, 2H), 3.74 (s, 2H), 3.25 (dt, 3J-
A
N
ACHTREUNG
1H), 2.60 ppm (dd, 2J
m/z: 293.06 [C13H16N4O2S]H+.
General procedure B: Re(CO)3 complex formation: One equivalent of
the ligand and one equivalent of [Re(CO)3Br3][NEt4]2 were added to a
N-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-thiophene-2-methylamine
(L5): As per general procedure A, with benzyl azide (138 mg, 1.0 mmol)
and 5 (157 mg, 1.0 mmol). The crude product was purified by column
chromatography with CH2Cl2 and MeOH (3%) to give a yellow oil
(131 mg, 46%). IR n˜ =2963, 1670, 1497, 1455, 1330, 1260, 1218, 1096,
1:1 mixture of methanol and water to form a 0.1 mm solution and stirred
at 658C. The reaction was followed by HPLC. After 3 h all of the starting
material had been consumed. The solvent was removed under reduced
pressure, and the residue redissolved in water. The product was purified
with a Sep-Pak column and eluted with a 1:2 ratio of water to methanol.
The fractions containing the product were combined and the solvent re-
moved under reduced pressure to give [Re(CO)3L] as a white powder.
1047, 798, 695 cmÀ1 1H NMR (CDCl3): d=7.38–7.36 (m, 4H), 7.29–7.25
;
(m, 2H), 7.21–7.19 (m, 1H), 6.95–6.91 (m, 2H), 5.51 (s, 2H), 4.01 (s, 2H),
3.92 (s, 2H), 1.57 ppm (bs, 1H); 13C NMR (CDCl3): d=ppm. HR-MS
(ES): m/z: 285.1171 [C15H16N4S]H+ (calcd. 285.1168).
S-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-2-(Boc-amino)ethanethiol
(L6a): As per general procedure A, with benzyl azide (346 mg,
2.60 mmol) and 6a (560 mg, 2.60 mmol). The crude product was purified
by column chromatography with CH2Cl2 and MeOH (5%) to give a
yellow oil (560 mg, 62%). IR n˜ =2976, 1698, 1508, 1456, 1391, 1365, 1341,
[Re(CO)3L1]: As per general procedure B, with L1 (9.5 mg, 0.04 mmol)
and [Re(CO)3Br3]
as a white powder (15 mg, 82%). IR n˜ =2923, 2022, 1902, 1867, 1633,
1074, 734 cmÀ1 1H NMR (CD3OD): d=7.97 (s, 1H), 7.49–7.33 (m, 5H),
5.88 (dd, 3J(H,H)=5.8 Hz, 2J
(H,H)=11.2 Hz, 1H), 5.64 (s, 2H), 5.20 (d
2J
(H,H)=11.2 Hz, 1H), 4.14–4.04 (m, 1H), 3.36–3.29 (m, 1H), 3.22 ppm
(dd, 2J(H,H)=17.7, 3J(H,H)=4.0 Hz, 1H); 13C NMR (CD3OD): d=
A
1270, 1250, 1165, 1048, 1029, 951, 743, 709 cmÀ1
;
1H NMR (CDCl3): d=
;
7.44 (s, 1H), 7.40–7.25 (m, 5H), 5.50 (s, 2H), 4.95 (bs, 1H), 3.79 (s, 2H),
A
ACHTREUNG
3.30 (m, 3J
U
G
AHCTREUNG
A
ACHTREUNG
198.3, 197.5, 196.7, 184.7, 144.1, 135.4, 130.2, 130.1, 129.6, 126.4, 56.0,
52.7, 27.5 ppm; HR MS (ES): m/z: 515.0370 [C15H12N4O5Re]À(calcd.
515.0371); elemental analysis (%) calcd. for C15H13N4O5Re: C 34.95, H
2.54, N 10.87; found: C 34.52, H 2.72, N 10.59.
S-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-2-(amino)ethanethiol
(L6):
L6a (460 mg, 1.32 mmol) was dissolved in CH2Cl2/TFA (9:1; 13 mL) and
stirred at room temperature overnight. The solvent was removed and the
crude product purified by column chromatography with CH2Cl2 and
MeOH (10%) to give a yellow oil (248 mg, 85%). IR n˜ =3036, 1673,
[Re(CO)3L2]: L2 (50 mg, 0.20 mmol) and [Re(CO)3Br3][NEt4]2 (157 mg,
R
0.20 mmol) were dissolved in a 1:1 mixture of methanol and water
(18 mL) and stirred at 658C for 3 h. HPLC analysis confirmed comple-
tion of the reaction. The solvent was removed under reduced pressure,
and the residue redissolved in methanol. The white precipitate was col-
lected by filtration, washed well with methanol and CH2Cl2 and dried
under vacuum (80 mg, 78%). IR n˜ =2021, 1921, 1893, 1864, 1658, 1615,
1457, 1434, 1200, 1179, 1129, 1057, 837.8, 799, 722 cmÀ1 1H NMR
;
(CDCl3): d=7.40 (s, 1H), 7.35–7.22 (m, 5H), 5.45 (s, 2H), 3.75 (s, 2H),
3.20 (t, 3J
N
N
(bs, 2H); 13C NMR (CDCl3): d=162.8, 145.2, 134.4, 129.2, 128.9, 128.2,
122.5, 54.3, 38.8, 28.8, 25.4 ppm; HR-MS (ES): m/z: 271.0983
[C12H16N4S]Na+ (calcd. 271.0988).
1363, 1343, 1128, 898, 764, 726, 717, 653, 644 cmÀ1
;
1H NMR
([D6]DMSO): d=8.41(s, 1H), 7.45–7.37 (m, 4H), 7.35–7.31(m, 1H),
N-Boc-S-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-cysteine methyl ester
(L7a): As per general procedure A, with benzyl azide (202 mg,
1.52 mmol) and 7a (419 mg, 1.52 mmol). The crude product was purified
by column chromatography with CH2Cl2 and MeOH (5%) to give a pale
yellow solid (544 mg, 88%). IR n˜ =3388, 3130, 2980, 2932, 2097, 1764,
1685, 1513, 1462, 1436, 1413, 1394, 1370, 1347, 1318, 1285, 1257, 1216,
1169, 1150, 1062, 1052, 1025, 989, 889, 862, 817, 783, 755, 732, 716,
2
2
ACHTREUNG
5.75 (s, 2H), 4.25 (d, J
3J(H,H)=4.8 Hz, 1H), 3.55 (dd, 2J
1H), 3.26 ppm (d, 2J(H,H)=16.9 Hz, 1H); 13C NMR ([D6]DMSO): d=
T
A
N
ACHTREUNG
AHCTREUNG
197.5, 197.0, 196.8, 179.2, 148.7, 134.5, 129.0, 128.7, 128.4, 123.6, 55.0,
54.3, 51.9 ppm; MS (ES): m/z: 517.03 [C15H13N4O5Re]H+; elemental anal-
ysis (%) calcd. for C15H13N4O5Re: C 34.95, H 2.54, N 10.87; found: C
34.91, H 2.54, N 10.73. Crystals suitable for X-ray diffraction were ob-
tained by diffusion of hexane into a solution of the complex in EtOH.
695 cmÀ1
2H), 5.41–5.36 (s, 1H), 4.55–4.51 (s, 1H), 3.80 (s, 2H), 3.73 (s, 3H), 2.98
(dd, 2J(H,H)=13.9 Hz, 3J(H,H)=5.1Hz, 1H), 2.90 (dd, 2J
(H,H)=
13.9 Hz, 3J(H,H)=5.8 Hz, 1H),1.43 ppm (s, 9H); 13C NMR (CDCl3): d=
;
1H NMR (CDCl3): d=7.41(s, 1H), 7.40–7.26 (m, 5H), 5.51(s,
A
N
ACHTREUNG
[Re(CO)3L3]NEt4: (36 mg, 0.13 mmol) was dissolved in a 1:1 mixture of
methanol and water (13 mL). The solution was neutralised with aqueous
ACHTREUNG
171.4, 145.3, 134.5, 129.2, 129.0, 128.2, 122.0, 54.4, 53.5, 52.7, 34.2, 28.5,
NEt4OH. [Re(CO)3Br3][NEt4]2 (100 mg, 0.13 mmol) was added and the
C
26.9 ppm; MS (ES): m/z: 408.12 [C19H26N4O4S]H+.
mixture stirred at 658C for 3 h. HPLC analysis confirmed the completion
of the reaction. The solvent was removed under reduced pressure, and
the residue redissolved in water. The crude product was purified with a
S-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-cysteine methyl ester (L7):
L7a (400 mg, 0.98 mmol) was dissolved in CH2Cl2/TFA (9:1; 10 mL) and
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6181

R. Schibli et al.
Sep-Pak column and eluted with a 1:2 ratio of water to methanol. The
fractions containing the product were combined and the solvent removed
under reduced pressure to give [Re(CO)3L3]NEt4 as a white powder
(50 mg, 57%). IR n˜ =3386, 2020, 1885, 1609, 1582, 1434, 1417, 1393, 1297,
5.1Hz, 3J
3.5 Hz, 3J(H,H)=1.0 Hz, 1H), 7.08 (dd, 4J
A
(H,H)=
G
ACHTREUNG
(s, 2H), 4.84–4.76 (m, 2H), 4.59–4.52 (m, 2H) 4.08–4.00 ppm (m, 1H);
13C NMR ([D6]acetone): d=197.7, 196.5, 192.3, 149.3, 139.2, 135.5, 130.3,
129.9, 129.6, 129.2, 128.2, 127.8, 123.3, 55.9, 55.6, 48.4 ppm; HR-MS (ES):
m/z: 555.0489 [C18H16N4O3ReS]+ (calcd. 555.0494); elemental analysis
(%) calcd. for C18H16N4O3ReS: C 34.07, H 2.54, N 8.83; found: C 33.96,
H 2.61, N 8.62.
1
1162, 1026, 1001, 900, 729, 698 cmÀ1; H NMR (CD3OD): d=7.95 (s, 1H),
3
7.41–7.38 (m, 4H), 5.64 (s, 2H), 3.54 (t, J
A
A
13C NMR (CD3OD): d=7.6, 27.0, 53.3, 55.9, 126.9, 129.6, 130.1, 130.2,
135.6, 147.3, 179.9, 197.8, 198.4 ppm; HR-MS (ES): m/z: 544.0739
[C16H11N3O7Re]À(calcd. 544.0160); elemental analysis (%) calcd. for
C24H31N4O7Re: C 42.79, H 4.64, N 8.32; found: C 41.31, H 4.99, N 8.00.
[Re(CO)3L6]Br: As per general procedure B, with L6 (78 mg,
0.33 mmol) and [Re(CO)3Br3][NEt4]2 (242 mg, 0.33 mmol). Analytically
U
pure [Re(CO)3L6]Br was obtained by washing the Sep-Pak purified
product with CH2Cl2 to remove traces of NEt4Br (65 mg, 40%). H NMR
1
[Re(CO)3L4]Br: As per general procedure B, with L4 (35 mg,
(CD3OD): d=8.18 (s, 1H), 7.45–7.41 (m, 5H), 5.70 (s, 2H), 5.26 (bs,
0.13 mmol) and [Re(CO)3Br3][NEt4]2 (97 mg, 0.13 mmol). The reaction
was carried out in methanol. [Re(CO)3L4]Br was isolated as a white
A
1H), 4.73 (bs, 1H), 4.43 (d, 2J
A
U
17.0 Hz, 1H), 2.98–2.84 (m, 3H), 2.13–2.02 ppm (m, 1H); 13C NMR
(CD3OD): d=194.6, 192.9, 192.6, 151.6, 135.0, 130.3, 130.25, 129.8, 124.8,
56.7, 44.2, 39.1, 34.2 ppm; MS (ES): m/z: 519.05 [C15H16N4O3ReS]+; ele-
mental analysis (%) calcd. for C15H16BrN4O3ReS: C 30.10, H 2.69, N
9.36; found: C 30.20, H 2.80, N 9.20. Crystals suitable for X-Ray diffrac-
tion were obtained by slow evaporation of a solution of the complex in
ethanol.
powder (51mg, 65%). IR n˜ =3014, 2873, 2023, 1906, 1494, 1447, 1154,
1112, 1052, 1044, 888, 772, 732, 696, 637, 626 cmÀ1 1H NMR (CD3OD):
;
d=8.77 (m, 4J
U
ACHTREUNG
7.8 Hz, 3J
G
(m, 4H), 7.12–7.09 (m, 2H), 5.60 (d, 2J
A
17.0 Hz, 1H) 4.48 (d, 2J
ACHTREUNG
16.6 Hz, 1H); 13C NMR (CD3OD): d=161.9, 153.3, 150.9, 141.4, 135.5,
130.1, 130.0, 129.0, 124.6, 124.2, 63.8, 56.2, 53.0 ppm (carbonyl carbons
not observed); HR-MS (ES): m/z: 550.0890 [C19H17N5O3Re]+ (calcd.
550.0884); elemental analysis (%) calcd. for C19H17BrN5O3Re: C 36.25, H
2.72, N 11.13; found: C, 36.66, H 2.92, N 10.68.
[Re(CO)3L7]Br: As per general procedure B, with L7 (9.5 mg,
0.04 mmol) and [Re(CO)3Br3][NEt4]2 (27 mg, 0.04 mmol). The reaction
C
was carried out in methanol. [Re(CO)3L7]Br was isolated as a white
powder (15 mg, 82%). 1H NMR (CD3OD): d=8.21(s, 1H), 7.46–7.40
(m, 5H), 5.73 (d, 2J
C
ACHTREUNG
ACHTREUNG
[Re(CO)3L5]Br: L5 (100 mg, 0.35 mmol) and [Re(CO)3Br3][NEt4]2
E
AHCTREUNG
(271mg, 0.35 mmol) were dissolved in methanol (35 mL) and stirred at
658C. A precipitate began to form after approximately 1hour. After 2 h
the reaction mixture was cooled to room temperature and the white pre-
cipitate collected by filtration, washed well with methanol and CH2Cl2
and dried under vacuum (120 mg, 54%). IR n˜ =2016, 1913, 1875, 1456,
14334, 1248, 1156, 1138, 1109, 1028, 991, 964, 820, 720, 701, 679, 660, 645,
(CD3OD): d=194.2, 192.5, 192.2, 170.9, 151.4, 135.0, 130.3, 130.2, 129.9,
125.0, 58.1, 56.8, 53.6, 39.9, 34.5 ppm; MS (ES): m/z: 577.01
[C17H18N4O5ReS]+.
[Re(CO)3L7b]Br: As per general procedure B, with L7b (46 mg,
0.16 mmol) and [Re(CO)3Br3]
A
633 cmÀ1
;
1H NMR ([D6]acetone): d=8.17 (s, 1H), 7.53 (dd, 4J
(H,H)=
A
(CO)3L7b]Br was isolated as a white powder (51mg, 58%). Compound
Table 2. Crystal structure parameters.
[Re(CO)3L2]
[Re(CO)3L3]NEt4
[Re(CO)3L6]Br
formula
Mr
C15H13N4O5Re
515.49
C49H64Cl2N8O14Re2
1432.38
C15H16BrN4O3ReS
598.49
size [mm]
description
system
space group
a [Š]
b [Š]
c [Š]
a [8]
b [8]
0.13”0.13”0.09
colourless block
monoclinic
P21/c
0.35”0.22”0.15
colourless prism
triclinic
0.45”0.17”0.04
colourless plate
monoclinic
I2/a
¯
P1
9.5972(1)
14.2008(2)
24.1727(2)
90
91.7106(9)
90
12.8499(15)
15.4486(13)
15.5010(14)
110.452(8)
92.469(8)
105.905(9)
2739.7(5)
2
20.3718(2)
6.59104(7)
28.9051(4)
90
105.3580(14)
90
g [8]
V [Š3]
Z
3292.98(6)
8
3742.53(7)
8
1c [gcmÀ3
]
2.080
1.736
2.124
m
A
]
7.415
4.582
8.762
F
A
1968
1420
2272
q range [8]
4
2.56 to 30.512.40 to 33.1 2.84 to 36.32
reflections measured
independent reflections
Rint
reflections observed
completeness [%]
40584
10048
0.0342
7409
360688
20900
0.0408
15711
100
58404
9054
0.0469
6991
100
99.9
rel. max. and min. transmission
data/restraints/parameters
goodness of fit on F2
final R indices [I>2s(I)]
1.0000 and 0.8493
10048/0/451
1.0000 and 0.6207
13602/0/684
1.0000 and 0.2186
9054/0/233
0.895
1.130
1.044
R1=0.0208, wR2=0.0323
R1=0.0374, wR2=0.0338
3 a0n.d809 and À0.8213.31
R1=0.0278, wR2=0.0813
R1=0.0417, wR2=0.0919
À1.313
R1=0.0260, wR2=0.0635
R1=0.0407, wR2=0.0683
2.082 and À3.612
R indices (all data)
residual electron [eŠ
À3
]
6182
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6173 – 6183

Incorporation of Metal Chelating Systems into Biomolecules
FULL PAPER
1
A: H NMR (CD3OD) d=8.02 (s, 1H), 7.46–7.39 (m, 5H), 5.89 (m, 1H),
[5] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113,
2056–2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
[6] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew.
Chem. 2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–
2599.
[7] T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, J. Am. Chem.
Soc. 2004, 126, 2853–2855.
[8] B. Colasson, M. Save, P. Milko, J. Roithova, D. Schroder, O. Rein-
aud, Org. Lett. 2007, 9, 4987–4990.
5.74 (d, 2J
2J
3.47–3.41(m, 1H), 3.28 (dd,
3.03 ppm (dd, 2J(H,H)=13.3 Hz, 3J
1H NMR (CD3OD) d=8.17 (s, 1H), 7.46–7.39 (m, 5H), 5.70
(m, 1H), 4.60 (m, 1H), 4.46 (d, 2J(H,H)=17.5 Hz, 1H), 4.24 (d, 2J-
(H,H)=17.5 Hz, 1H), 3.32–3.31 (m, 1H), 2.88 (dd, 2J(H,H)=14.2 Hz, 3J-
(H,H)=11.4 Hz, 1H), 2.50 ppm (m, 1H); 13C NMR (CD3OD): d=194.9,
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
T
AHCTREUNG
A
N
ACHTREUNG
193.1, 193.0, 152.1, 150.9, 135.1, 135.0, 130.4, 130.3, 130.2, 129.9, 129.8,
125.3, 124.5, 56.7, 43.0, 41.3, 34.5, 34.1 ppm; MS (ES): m/z: 562.77
[C16H16N4O5ReS]+; elemental analysis (%) calcd. for C16H15N4O5ReS: C
34.22, H 2.69, N 9.98; found: C 34.38, H 2.94, N 9.82.
[9] Q. Dai, W. Z. Gao, D. Liu, L. M. Kapes, X. M. Zhang, J. Org. Chem.
2006, 71, 3928–3934.
[10] R. J. Detz, S. A. Heras, R. de Gelder, P. van Leeuwen, H. Hiemstra,
J. N. H. Reek, J. H. van Maarseveen, Org. Lett. 2006, 8, 3227–3230.
[11] R. M. Meudtner, R. Ostermeier, M. Goddard, C. Limberg, S. Hecht,
Eur. J. Inorg. Chem. 2007, 13, 9834–9840.
[12] U. Monkowius, S. Ritter, B. Kçnig, M. Zabel, H. Yersin, Eur. J.
Inorg. Chem. 2007, 4597–4606.
[13] T. L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg,
V. Maes, D. Tourwe, R. Schibli, J. Am. Chem. Soc. 2006, 128, 15096–
15097.
[14] S. R. Banerjee, M. K. Levadala, N. Lazarova, L. Wei, J. F. Valliant,
K. A. Stephenson, J. W. Babich, K. P. Maresca, J. Zubieta, Inorg.
Chem. 2002, 41, 6417–6425.
[15] N. Lazarova, J. Babich, J. Valliant, P. Schaffer, S. James, J. Zubieta,
Inorg. Chem. 2005, 44, 6763–6770.
[16] J. K. Pak, P. Benny, B. Spingler, K. Ortner, R. Alberto, Chem. Eur.
J. 2003, 9, 2053–2061.
[17] D. R. van Staveren, P. D. Benny, R. Waibel, P. Kurz, J.-K. Pak, R.
Alberto, Helv. Chim. Acta 2005, 88, 447–460.
X-ray crystallography: Crystallographic data were collected at 183(2) K
by using an Oxford Diffraction Xcalibur system with a Ruby detector
(MoKa radiation, l=0.7107 Š) by using graphite-monochromated radia-
tion. Suitable crystals were covered with oil (Infineum V8512, formerly
known as Paratone N), mounted on top of a glass fibre and immediately
transferred to the diffractometer. The program suite CrysAlisPro was used
for data collection, semi-empirical absorption correction and data reduc-
tion.[28] Structures were solved with direct methods using SIR97[29] and
were refined by full-matrix least-squares methods on F2 with SHELXL-
97.[30] The structures were checked for higher symmetry with the help of
the program Platon.[31] CCDC 680569 ([Re(CO)3L2]), 680568 ([Re-
(CO)3L3]NEt4) and 680570 ([Re(CO)3L6]Br) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. The crystal structure parameters
can be found in Table 2.
[18] R. Schibli, R. La Bella, R. Alberto, E. Garcia-Garayoa, K. Ortner,
U. Abram, P. A. Schubiger, Bioconjugate Chem. 2000, 11, 345–351.
[19] N. Marti, B. Spingler, F. Breher, R. Schibli, Inorg. Chem. 2005, 44,
6082–6091.
[20] O. Karagiorgou, G. Patsis, M. Pelecanou, C. P. Raptopoulou, A.
Terzis, T. Siatra-Papastaikoudi, R. Alberto, I. Pirmettis, M. Papado-
poulos, Inorg. Chem. 2005, 44, 4118–4120.
Labelling
(OH2)3(CO)3]+ was prepared according to the literature procedure.[32]
99mTcO4]À (1mL) in NaCl (0.9%) was added to the IsoLink(TM) kit
(Mallinckrodt–Tyco, Petten, Holland) through a septum. The reaction
was heated for 20 min at 1008C. The solution was cooled and neutralised
to pH 7.2 with a mixture of HCl (1m) and phosphate buffer (1m, pH 7.4).
with
[
99 mTc
(OH2)3(CO)3]+:
The
precursor
[
99mTc-
ACHTREUNG
[
Stock solutions (10À2 to 10À7 m) of ligands L1–L8 were prepared in PBS,
[21] R. F. Vitor, S. Alves, J. D. G. Correia, A. Paulo, I. Santos, J. Organo-
met. Chem. 2004, 689, 4764–4774.
pH 7.4. A solution of [99mTc(CO)3
A
added to the relevant ligand (50 mL) diluted with PBS (400 mL, pH 7.4)
to give final concentrations between 10À3 and 10À8 m. The reaction mix-
tures were heated for 60 min at 1008C. Radiolabelling yields were deter-
mined by HPLC. The identity of the products was confirmed by compari-
son of the g-traces of the 99mTc complexes with the UV traces of the cor-
responding Re complexes.
[22] L. J. Farrugia, J. Appl. Crystallogr. 1997, 30, 565.
[23] L. V. Lee, M. L. Mitchell, S. J. Huang, V. V. Fokin, K. B. Sharpless,
C. H. Wong, J. Am. Chem. Soc. 2003, 125, 9588–9589.
[24] B. Munchpetersen, L. Cloos, H. K. Jensen in Advances in Enzyme
Regulation, Vol. 35, 1995, pp. 69–89.
[25] S. Celen, T. J. De Groot, J. Balzarini, K. Vunckx, C. Terwinghe, P.
Vermaelen, L. Van Berckelaer, H. Vanbilloen, J. Nuyts, L. Mortel-
mans, A. Verbruggen, G. Bormans, Nucl. Med. Biol. 2007, 34, 283–
291.
[26] K. K. Ogilvie, Can. J. Chem. 1973, 51, 3799–3807.
[27] R. Alberto, A. Egli, U. Abram, K. Hegetschweiler, V. Gramlich,
P. A. Schubiger, J. Chem. Soc. Dalton Trans 1994, 2815–2820.
[28] CrysAlisPro Software system; Oxford Diffraction Ltd., vers. 171.32
Oxford, UK, 2007.
[29] A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giaco-
vazzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori, R. Spagna, J.
Appl. Crystallogr. 1999, 32, 115–119.
Acknowledgements
We are very grateful to Judith Stahel for help with the radiolabelling ex-
periments and to Alain Blanc and Simone Jeger for measuring MS. We
thank Dr Dominique Desbouis for helpful advice regarding the thymi-
dine experiments, and Mallinckrodt–Tyco, Petten for financial support.
[1] R. A. Ferrieri in Handbook of Radiopharmaceuticals: Radiochemis-
try and Applications, (Eds.: M. J. Welch, C. S. Redvanly), John Wiley
& Sons, 2003, pp. 229–282.
[2] M. T. Madsen, J. Nucl. Med. 2007, 48, 661–673.
[3] S. R. Banerjee, K. P. Maresca, L. Francesconi, J. Valliant, J. W.
Babich, J. Zubieta, Nucl. Med. Biol. 2005, 32, 1–20.
[4] S. R. Banerjee, J. W. Babich, J. Zubieta, Chem. Commun. 2005,
1784–1786.
[30] G. M. Sheldrick, Acta Cryst. 2008, A64, 112–122.
[31] A. L. Spek, J. Appl. Crystallogr. 2003, 36, 7–1 3.
[32] R. Alberto, K. Ortner, N. Wheatley, R. Schibli, A. P. Schubiger, J.
Am. Chem. Soc. 2001, 123, 3135–3136.
Received: December 21, 2007
Published online: May 21, 2008
Chem. Eur. J. 2008, 14, 6173 – 6183
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6183