A Click procedure with heterogeneous copper to tether technetium-99m chelating agents and rhenium complexes. Evaluation of the chelating properties and biodistribution of the new radiolabelled glucose conjugates

Article abstract of DOI:10.1016/j.carres.2010.10.011

An efficient protocol was developed to tether chelating agents and rhenium complexes onto a glucoside scaffold with a heterogeneous copper catalyst via click chemistry. The supported catalyst avoids the formation of unwanted copper complexes during the cyclisation step. The possibility to graft a pre-chelated M(CO)₃ core by click chemistry onto a biomolecule was highlighted for the first time. ^99mTc(CO)₃-glucoconjugates displayed excellent in vitro stability, a fast in vivo blood clearance and a low specific organ uptake or long-term retention in spleen and stomach.

Full text of DOI:10.1016/j.carres.2010.10.011

Carbohydrate Research 346 (2011) 26–34
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A Click procedure with heterogeneous copper to tether technetium-99m
chelating agents and rhenium complexes. Evaluation of the chelating
properties and biodistribution of the new radiolabelled glucose conjugates
Eric Benoist a,b, , Yvon Coulais c, Mehdi Almant d, José Kovensky d, Vincent Moreau d, David Lesur d,
⇑
Marine Artigau a,b, Claude Picard a,b, Chantal Galaup a,b, Sébastien G. Gouin d, ,
⇑
a Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, SPCMIB, UMR 5068, 118, route de Narbonne, F-31062 Toulouse cedex 9, France
b Université de Toulouse;UPS;Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, SPCMIB, UMR 5068, 118, route de Narbonne,
F-31062 Toulouse cedex 9, France
c Laboratoire ‘Traceurs et traitement de l’image’, Faculté de Médecine de Purpan, EA 3033, 133, route de Narbonne, 31062 Toulouse cedex 9, France
d Laboratoire des Glucides UMR CNRS 6219, Institut de Chimie de Picardie, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2010
Received in revised form 7 October 2010
Accepted 10 October 2010
Available online 20 October 2010
An efficient protocol was developed to tether chelating agents and rhenium complexes onto a glucoside
scaffold with a heterogeneous copper catalyst via click chemistry. The supported catalyst avoids the for-
mation of unwanted copper complexes during the cyclisation step. The possibility to graft a pre-chelated
M(CO)3 core by click chemistry onto a biomolecule was highlighted for the first time. 99mTc(CO)3-gluco-
conjugates displayed excellent in vitro stability, a fast in vivo blood clearance and a low specific organ
uptake or long-term retention in spleen and stomach.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Glycoconjugates
Heterogeneous click chemistry
Bioconjugation
Metal complexes
1. Introduction
by the short half life of the positron emitting fluorine-18
(T1/2 = 109.8 min), which should be produced on site by expensive
Carbohydrates play a pivotal role in a host of biological events
including molecular recognition, inflammation, tumour metastasis
and viral or bacterial adhesion.1 In particular, they provide a major
energy source for life forms through the processing of glucose in-
side the cells. The energy requirement in many tumour types is sig-
nificantly higher than in normal cells due to a rapid growing and
altered metabolism. This feature has been extensively explored
for the development of glucose conjugates as selective radiotracer
cyclotrons.
In this regard, the development of more readily available FDG
analogues would be of high interest. The technetium-99 m radio-
nuclide (99mTc) is extensively used in nuclear imaging with single
photon emission computed tomography (SPECT), due to ideal
imaging characteristics (140 keV
c emitter, relatively short half life
of 6 h) combined with a convenient availability at low cost from
the commercial 99Mo/99mTc generator. A wide range of chelators
is now available to efficiently coordinate diverse technetium spe-
for tumours in oncology. FDG (2-deoxy-2-[18F]fluoro-
D-glucose)
cies like TcO3+ and TcðCOÞ3 cores, and to form stable complexes
þ
has become an important radiopharmaceutical approved by the
Food and Drug Administration to detect melanoma, lymphoma,
breast and lung cancers by positron emission tomography (PET)
scans.2 FDG is internalized into the cells by the GLUT1 transporter
and phosphorylated by hexokinase to a very polar product that
cannot diffuse out of the cell. Applications are however limited
under physiological conditions.3 Numerous chemical strategies
have been developed to graft such compounds onto biologically
relevant carriers such as peptides,4 antibodies5 and carbohy-
drates.6,7 In particular, several groups recently reported the syn-
thesis of glucose derivatives containing 99mTc-chelating systems
to substitute the expensive FDG in the localization of tumours.8
These complexes were chemically grafted onto the different hydro-
xyl groups of the glucose scaffolds by glycosylation (C-1),9 nucleo-
philic substitutions and reductive aminations (C-2, 3, 6),10 or
peptidic coupling when starting from glucose amine.11 Despite
the numerous attempts, a 99mTc based radiopharmaceutical that
fulfils the criteria of a potent [18F]-FDG surrogate has not been
⇑
Corresponding authors.
E-mail addresses: benoist@chimie.ups-tlse.fr (E. Benoist), Sebastien.Gouin@univ-
nantes.fr (S.G. Gouin).
Present address: Université de Nantes, CEISAM, Chimie Et Interdisciplinarité,
Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR des Sciences et des
Techniques, 2, rue de la Houssinière, BP 92208, 44322 NANTES Cedex 3, France.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.10.011

E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
27
designed yet. It seems that the major drawback of this strategy is
the presence of the technetium complex, which is too hindered
to allow the glycoconjugate internalization into cells via the GLUT1
transporter.12
co-workers reported that a 1,2,3-triazole ring formed during the
cyclisation step is an effective binder for M(CO3)+ core.16 Following
their so-called ‘click to chelate’ approach, we synthesized pre-che-
lator 417 that will potentially bind the metallic core through both
nitrogen atoms and the formed triazole.
Nevertheless, carbohydrates attract much attention, as hydro-
philic and biocompatible scaffolds for the development of molecu-
lar imaging agents,13 or as vectors able to target carbohydrate
binding proteins (lectins).14 In this regard, the development of
simple and efficient protocols to graft chelating agents or metal
complexes onto carbohydrate scaffolds are of particular interest.
In the present work we described a simple and efficient protocol
to synthesize C-1 functionalized glucose derivatives-containing tri-
dentate chelating systems via a copper catalysed azide–alkyne
cyclization reaction (click reaction). We demonstrated for the first
time the possibility to graft a pre-chelated M(CO)3 core directly
onto a biomolecule by click chemistry. The preparation and spec-
troscopic analysis of both cold Re(CO)3 and radioactive 99mTc(CO)3
glycoconjugates are reported. In vitro histidine challenge experi-
ments and first biodistributions of technetium glucoconjugates
are also investigated.
The chelate systems 1 and 3 were prepared in excellent yields
(near 90%) by refluxing commercial di-(2-picolyl)amine or tert-bu-
tyl iminodiacetate with propargyl bromide in the presence of
potassium carbonate in acetonitrile as previously described.18 In
a similar way, the reaction of propargyl amine with the chlorohy-
drate of 2-chloroquinoline gave the desired compound 2 in a fair
yield of 62%. Particular interest arises in developing a fast access
to such chelating systems, which can be either used to design
radioimaging or fluorescent probes with technetium or rhenium,
respectively. N-Propargyl-di-(2-picolyl)amine 4 was recently pre-
pared by reductive amination with a moderate yield of 57%.19 Con-
sequently, we developed a more suitable synthetic pathway to
prepare this compound in gram-scale starting from 2-(amino-
methyl)pyridine, as outlined in Scheme 1. Following a three-step
sequence already described for analogous derivatives,20 the amine
was first protected as a tert-Boc carbamate to avoid the formation
of N,N-dipropargyl derivatives during the alkylation step. Subse-
quent treatment with an excess of sodium hydride in the presence
of propargyl bromide, followed by amine deprotection with TFA,
provided 4 with an overall yield of 89%.
2. Results and discussion
2.1. Synthesis of glucoconjugates and their corresponding
metallic complexes (M = 99mTc, Re)
As proof of concept, two tricarbonylrhenium complexes were
readily obtained by reacting equivalent amounts of ligands 1 or 2
and Re(CO)5Br in refluxing methanol. The co-ordination reactions
were quantitative in 6 h and after purification led to mononuclear
complexes isolated as bromine salts of general formula [Re(CO)3(L)]
[Br].
An ideal bifunctional chelating agent should (i) coordinate a
metal ion with a high yield, (ii) form metal complexes with both
high thermodynamic stability and in vivo kinetic inertness to min-
imize metal toxicity, (iii) be produced quickly, in a gram-scale and
with an excellent overall yield. For the complexation of the
M(CO3)+ core, recent in vitro and in vivo investigations showed
that tridentate chelating systems are more appropriate.15 A Tc-
or Re-tricarbonyl complex with a tridentate ligand is less prone
to cross-metallation due to its coordinative saturation and thermo-
dynamic stability. Among these ligands, iminodiacetic acid (IDA)
and di-(2-picolyl)amine (DPA) derivatives react avidly with the
fac-[M(CO)3]+ core to form complexes with a stable octahedral
coordination sphere. We first designed a set of alkynyl-armed che-
lating systems 1–4 based on DPA, IDA or analogous derivatives
(Chart 1). Building blocks 1–3 could be considered as bifunctional
chelating agents where the acetylenic arm allows conjugation to
the glucopyranosyl azide by click chemistry. Recently, Schibli and
The glucopyranosyl azide21 scaffold was easily obtained in two
steps from glucose pentaacetate22 (Scheme 2). Displacement of the
anomeric acetate group with TMSN3 in the presence of tin chloride
followed by deprotection under Zemplén conditions (NaOMe in
MeOH) led to 5 in 70% yield. The first copper(I)-catalysed cycload-
dition with alkynyl-armed chelating agent 1 was performed in the
presence of copper sulfate and sodium ascorbate in a mixture of
tert-butanol and water to ensure the solubility of reagents. The
reaction proceeded smoothly, leading to the expected cycloadduct
6 polluted by its copper complex, which was identified by ESI-MS
during the experiment.
It was previously reported that treatment by a chelating resin
(QuadraPure-IDA) allows the regeneration of the desired cycload-
ducts by transchelation of copper.16i However, this procedure will
be ineffective for chelators possessing high kinetic inertness or
stronger binding constants than the iminodiacetic groups attached
to the resin. Performing the cyclization step with a supported or
heterogenous source of copper(I) would be more appealing with
this regard. Lipshutz and Taft have developed an efficient catalyst
for the Huisgen [3+2] cyclization based on copper impregnated
wood charcoal.23 Interestingly, the authors claimed that the reac-
tion takes place heterogeneously without any leaching of copper
R
R = pyr, 1
N
R = qui, 2
N
R
NH
R = COOtBu, 3
pre-chelator 4
Tridentate chelating agents 1-3
Chart 1. Design features of Tc-chelating agents.
Br
, NaH, THF
(Boc)2O, CH2Cl2
N
N
N
99%
NH2
95%
NHBoc
NBoc
i) TFA, CH2Cl2
ii) NaOH
N
95%
NH
4
Scheme 1. Synthetic pathway for the preparation of 4.

28
E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
N
N
OH
OAc
N
1
i) TMSN3, SnCl4
HO
AcO
AcO
O
O
ii) MeONa, MeOH
HO
N3
1.1 eq CuSO4,
2.2 eq NaAsc,
20 min
OAc
HO
5
AcO
70% yield for 2 steps
2+
N
N
N
OH
Cu
OH
N
N
HO
HO
N
N
N
N
HO
HO
N
N
N
O
O
HO
HO
6
Scheme 2. Synthetic pathway for the preparation of glycoconjugate 6.
in solution as indicated by quantitative ICP-AES performed on
crude products. Initial reaction trials with our substrates were dis-
appointing as no traces of cycloadducts were observed after several
days at room temperature. Earlier studies have shown that micro-
wave irradiation could significantly improve the copper(I)-cata-
lysed azide–alkyne cyclization (CuAAC) reaction kinetics
compared to classical heating.24 We were delighted to see that un-
der microwave irradiation at 100 °C for 15 min, the expected glyco-
conjugate 6 could be isolated as a pure compound in 91% yield,
after a simple filtration of the catalyst (Table 1). Additionally,
ESI-MS analysis revealed that no detectable copper complex was
present in the medium. The heterogeneous CuAAC was also highly
regioselective yielding 1,4-disubstituted-1,2,3-triazoles identified
Compounds were fully characterized by classical spectroscopic
methods. Briefly, the NMR spectra revealed the characteristic sig-
nals of the sugar unit and the ligand or the chelating moiety. Due
to the electronic influence of the tricarbonyl rhenium core, a signif-
icant downfield shift of the hydrogen resonances near the chelat-
ing N-atoms was observed. In such complexes, the protons of the
methylene groups adjacent to the aromatic rings are non-equiva-
lent. The 13C NMR spectra of 6-Re and 8-Re show only two peaks
near 195–197 ppm with the characteristic ratio of peak height of
2:1 for the fac-[Re(CO)3]+ core indicating that (i) two CO groups
are magnetically equivalent, (ii) the coordination sphere exhibits
mirror symmetry including one of the three facial carbonyl groups
and the coordinated amino group, as previously reported for such
chelating units.27 Interestingly, the complex 7-Re exhibited differ-
ent analytical features. Firstly, the reaction of glycoconjugate 7
with Re(CO)5Cl in refluxing methanol, led to a mixture of two
structural isomers in a 1:1 ratio. The observation of two diastereo-
isomers can only result from the formation of a complex where the
ligand is bound in a bidentate fashion. This result is surprising as
Schibli and co-workers having demonstrated recently that this
kind of ligand acted as a tridentate chelate.12 Unfortunately, it
was not possible by HPLC to achieve isomeric resolution under a
wide range of chromatographic conditions. 2D-NMR experiments
suggested that the rhenium core was coordinated by the (2-amino-
methyl)pyridine part of the glycoconjugate, giving a neutral com-
plex of the general formula [ReCl(CO)3(7)]. We hypothesized that
the nitrogens on the triazole ring may be deactivated due to the
electroattractive effect of the endocyclic oxygen atom.
by the large
D
(dC-4–dC-5) values (14–23 ppm) observed in 13C
NMR for the different structures.25 This procedure has been suc-
cessfully applied to pre-chelator 4, allowing conjugation and for-
mation of the additional chelating group, simultaneously. When
the reaction was conducted without catalyst, we obtained a mix-
ture of 1,4- and 1,5-disubstituted-1,2,3-triazoles. Interestingly,
the tridentate complex 1-Re containing the Re(CO)3 core can be di-
rectly introduced onto the carbohydrate scaffold, leading to 6-Re
with a moderate yield of 35% after HPLC purification. To our knowl-
edge, this is the first example of a pre-chelated M(CO)3 core di-
rectly grafted onto a biomolecule by click chemistry. This finding
is of interest considering the existence of two radioactive isotopes
of rhenium emitting both b-radiations that are able to kill tumour-
al cells
(
186Re: T1/2 = 90 h, E(bꢀ) = 1.07 MeV, 188Re T1/2 = 17 h,
E(bꢀ) = 2.12 MeV). Additionally, rhenium exhibits very similar
chemistry to technetium, and we expect that this reaction could
also occur with this metal. A direct introduction of a pre-chelated
Re or Tc core onto a biomolecule may be an alternative to the cur-
rent labelling strategies. Therefore, the present procedure, can be
considered as an extension of the ‘click to chelate’ concept de-
scribed by Schibli for the synthesis of 1,2,3-triazole chelators and
their in situ radiolabelling with technetium.16
2.2. In vitro and in vivo studies
The 99mTc-complexes were prepared by mixing the precursor
fac-[99mTc(CO)3(H2O)3]+ synthesized using the Isolink kit (Mal-
linckrodt Inc.), with compounds 6, 7, 8 or 10 in a methanol/buffer
solution pH 3.4. After 30 min at 95 °C, [99mTc(CO)3(H2O)3]+ was en-
tirely consumed and a new product was observed on the radio-
chromatogram. The four radioconjugates were obtained in an
excellent radiolabelling yield ranging from 95% for 6-Tc and 7-Tc,
to 90% for 8-Tc and 10-Tc, respectively. As expected, the radiola-
belling of 7 led to a mixture of two isomers in an approximate
1:1 ratio. To characterize the pure radiolabelled glycocomplexes,
HPLC co-injections of 99mTc-radiocomplexes with the correspond-
ing ‘cold’ rhenium complexes were performed. In all cases, the
retention times of the complexes were analogous, as illustrated
in Figure 1 for 6-Tc and 6-Re. These results evidenced that the
structures of the formed complexes are similar for technetium at
the tracer level or with rhenium at higher metal concentration.
Copper sulfate and sodium ascorbate26 were a more efficient
catalyst system for grafting 2 and 2-Re onto the glucoside scaffold.
It is noteworthy that glycoconjugate 8-Re may be a potential fluo-
rescent imaging agent for in vitro investigations of cellular uptake
as suggested by a previous report of Zubieta and co-workers with a
similar rhenium carbohydrate complex.27
Surprisingly, the tert-butyl esters of product 9 were stable to
acidic conditions and traces of these esters remain even after
heating the mixture at 100 °C in pure trifluoroacetic acid. In con-
trast, saponification with NaOH followed by acidification with di-
luted HCl allowed
a quantitative isolation of the expected
product 10.28

E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
29
Table 1
Copper catalysed formation of the glycoconjugates and glycocomplexes
Entry
Chelator/complex
Glycoconjugate/glycocomplex
Catalyst
C/Cu
Time
Yield (%)
N
OH
N
1
15 min
91b
N
HO
HO
N
N
N
O
HO
OH
6
N
N
H
4
H
N
N
N
N
HO
HO
N
2
3
C/Cu
C/Cu
15 min
87b
O
HO
7
OH
N
N
N
N
N
HO
HO
N
N
+
CO
CO
CO
CO
+
35c
O
8 h
Re
Re
1-Re
N
N
HO
CO
CO
6-Re
N
N
N
N
4
OH
CuSO4/NaAsc
15 min
70b
N
N
N
HO
HO
N
N
N
O
2
8
HO
OH
N
N
HO
HO
N
N
N
N
CO
CO
+
CO
CO
O
+
5
6
CuSO4/NaAsc
45 min
26c
Re
Re
N
HO
OH
N
CO
CO
2-Re
8-Re
CO2R
CO2R
CO2tBu
CO2tBu
N
N
HO
HO
N
N
N
O
CuSO4/NaAsc
30 min
Quantb
3
HO
9 R=tBu
10 R=H
a
a
NaOH/EtOH then HCl (99% yield).
Based on the crude material.
After isolation by preparative HPLC.
b
c
High stability of the metal complexes toward competitive
isomers is detrimental for in vivo administration since the blood
clearance and radiopharmaceutical biodistribution may be altered.
Nevertheless, in this first assay, complex 7-Tc was only used as a
reference to compare the biological behaviours of this bidentate
glycocomplex versus a tridentate complex (6-Tc, 10-Tc). The re-
sults are expressed as percent of injected dose per organ (%ID/or-
gan) at 5, 30 and 240 min post-injection, as shown in Table 2.
Complex 6-Tc shows a high liver uptake (from 31% ID at 5 min
to 18% ID at 240 min) and is preferentially eliminated by the hepa-
tobiliary system, as revealed by the 45% ID at 240 min post-injec-
tion. Its elimination via the renal urinary excretion route is not
significant (2% ID at 60 min) and after 240 min post-injection only
11.2% ID was detected in the urine. In contrast, complexes 7-Tc and
10-Tc exhibit modest hepatobiliary elimination and were mainly
excreted via the renal pathway. Thus, complex 10-Tc presented
the highest concentration of radioactivity in urine at 240 min
(36% ID), indicating its hydrophilic character, as expected for an
anionic complex. More interestingly, the rapid clearance of these
compounds from the blood-stream, reflected by the low blood
ligands is of primary importance for the development of biologi-
cally relevant imaging probes. In vitro stability of the radiolabelled
glycocomplexes was assessed over a 24 h period with a 250-fold
molar excess of histidine, a high affinity competitive ligand for
the [99mTc(CO)3]+ core.29 No significant differences were observed
for the tridentate chelates 6-Tc, 8-Tc, 10-Tc and 7-Tc. Less than
10% of 99mTc was dissociated from the complexes after 24 h incu-
bation (data not shown). These results showed that the inertness
of the tridentate Tc-complexes towards transchelation, and are
consistent with previously reported histidine challenge assays
showing the stability of DPA and IDA-frameworks.30 They also con-
firm that the sixth coordination site of the bidentate complex 7-Tc
was occupied by a chlorine; a water molecule would have led to a
higher exchange rate.31
Biodistribution studies in healthy male Wistar rats were per-
formed to evaluate the organ uptakes and clearances of the
99mTc(CO)3-complexes 6-Tc, 10-Tc and 7-Tc (mixture of two iso-
mers). It is well known that the formation of metal complex

30
E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
Figure 1. HPLC profiles of 6-Tc (up, radiometric) and 6-Re (bottom, UV/230 nm) (elution conditions: MN Nucleodur C18 ec column 125 ꢁ 4 mm, 5
lm; eluent: 0.1% TFA in
H2O/MeOH 60:40).
Table 2
Biodistributions of 6-Tc, 7-Tc and 10-Tc in rats, at various time post-injection (the results are the average of at least three experiments)
Organ
%ID/organ
7-Tc
6-Tc
30 min
10-Tc
30 min
5 min
240 min
5 min
30 min
240 min
5 min
240 min
Carcass
Liver
Intestine
Stomach
Pancreas
Heart
32.03 1.82
30.81 2.70
17.42 6.62
3.04 2.29
2.46 0.03
0.11 0.00
0.39 0.06
8.22 3.64
0.06 0.01
0.03 0.01
4.99 0.43
15.23 7.06
26.26 2.13
35.68 6.54
2.90 1.36
1.89 0.08
0.05 0.02
0.18 0.07
2.05 0.71
0.08 0.03
0.04 0.01
1.35 0.69
5.07 1.51
17.67 3.06
45.21 12.52
3.07 1.52
1.60 0.32
0.02 0.01
0.09 0.02
0.89 0.40
0.02 0.01
0.01 0.01
0.26 0.11
54.04 1.52
11.54 1.50
4.06 0.34
0.63 0.04
0.95 0.14
0.30 0.09
0.69 0.12
14.71 2.37
0.17 0.01
0.05 0.01
13.24 0.25
25.26 2.44
19.76 1.47
3.91 1.13
1.33 0.72
1.68 0.13
0.10 0.02
0.29 0.01
5.46 0.56
0.07 0.01
0.02 0.00
5.04 1.01
8.25 3.41
20.56 0.85
10.04 1.50
0.99 0.19
2.03 0.64
0.02 0.01
0.08 0.04
1.64 0.11
0.05 0.01
0.01 0.00
0.65 0.37
50.23 1.85
8.15 0.67
4.61 0.92
0.67 0.14
0.75 0.01
0.30 0.01
0.71 0.15
13.24 5.58
0.13 0.01
0.04 0.03
16.25 1.40
34.72 5.61
3.82 0.44
10.58 2.83
2.82 2.46
0.40 0.07
0.13 0.04
0.33 0.08
7.39 2.55
0.05 0.02
0.01 0.01
6.01 0.28
5.36 1.29
1.68 0.26
12.88 8.54
3.93 1.61
0.17 0.04
0.03 0.01
0.07 0.02
1.18 0.24
0.03 0.01
0.02 0.01
0.86 0.13
Lungs
Kidneys
Spleen
Brain
Blood
activity, indicated their high stability against metal exchange reac-
tions with blood proteins. Additionally, the spleen and stomach
values were low, indicating minimal in vivo oxidation of techne-
tium to pertechnetate or reduction to colloidal Tc. Altogether, these
results highlight that the new glycoconjugates are able to shield
and stabilize 99mTc in vivo. In addition, the only significant differ-
ence between the tridentate complexes 6-Tc and 10-Tc and the
bidentate one concerns the liver uptake. No specific uptake or
long-term retention was observed in other organs or tissues for
complexes 6-Tc and 10-Tc. In contrast, 7-Tc exhibited a high liver
uptake after 240 min, as illustrated by the 21% ID at 240 min post-
injection.
3. Experimental
3.1. General methods
All purchased materials were used without further purification.
Methylene chloride was distilled from calcium hydride and tetra-
hydrofuran over sodium and benzophenone. Analytical thin layer
chromatography (TLC) was carried out on Merck D.C.-Alufolien
Kieselgel 60 F254. Flash chromatography (FC) was performed on
GEDURAN SI 60, 0.040–0.060 mm pore size using distilled solvents.
N-Propargyl-di-(2-picolyl)amine 1 and 3-[bis(tert-butoxycarbon-
ylmethyl)amino]-prop-1-yne 3 were prepared as described previ-
ously.18 Re(CO)5Br was purchased from Aldrich Chemical
Company; 1H, and 13C nuclear magnetic resonance (NMR) spectra
were recorded at 300 (75.5) MHz with a Bruker AC-300 spectrom-
eter or at 500 (125) MHz with a Bruker Avance DRX 500, and
chemical shifts are reported in parts per million relative to tetra-
methylsilane or a residual solvent peak (CHCl3: 1H: d 7.26, 13C: d
77.2). Peak multiplicity is reported as: singlet (s), doublet (d), trip-
let (t), quartet (q), pentet (p), sextet (s), multiplet (m) and broad
(br). Mass spectra were obtained on a Perkin–Elmer Sciex API
365 (electrospray), a DSQ2 Thermofisher (chemical ionisation) or
a Nermag 10-10 (FAB) mass spectrometer. High Resolution Mass
Spectra (HRMS) were obtained by Electrospray Ionisation (ESI) on
a Micromass-Waters Q-TOF Ultima Global. Optical rotations were
measured on a 343 Perkin–Elmer automatic polarimeter at 20 °C
2.3. Conclusion
We have developed an efficient protocol to graft 99mTc-chelat-
ing agents and rhenium complexes onto a glucose scaffold.
99mTc(CO)3-complexes 6-Tc and 10-Tc displayed in vitro stability
against histidine exchange reactions, a fast blood clearance, and
low specific uptake or long-term retention in spleen and stomach
revealing a high in vivo stability. More interestingly, the feasibility
to directly tether a pre-chelated Re(CO)3 core by click chemistry
was highlighted for the first time. The heterogeneous cyclization
protocol developed may also be applicable to other metallic cores
and more complex carbohydrate scaffolds. Work in this direction
is currently being performed in our group.

E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
31
in a 1 cm cell in the stated solvent; [
a]
values are given in
3.4. N-Boc,N-propargyl-2-picolylamine
D
10ꢀ1 deg cm2 gꢀ1 (concentration, c, given as g/100 mL). Microwave
irradiation was performed in a CEM Discover apparatus (300 W).
To a solution of N-Boc-2-picolylamine (11.3 g, 54.3 mmol) in
freshly distilled THF (200 mL) at 0 °C, sodium hydride (5.2 g,
217 mmol) was added gently in portions. The solution was stirred
for 20 min and treated dropwise with propargyl bromide (80 wt %
in toluene, 7.9 mL, 70.6 mmol). After stirring for 3 h at room tem-
perature, the solution was quenched cautiously with water
(100 mL). The aqueous layer was extracted twice with CH2Cl2
(200 mL). The combined extracts were washed with brine (3ꢁ)
and dried over Na2SO4. The crude product was purified by chroma-
tography on silica gel (CH2Cl2–MeOH 98:2). The product was iso-
lated as a pale yellow oil (12.7 g, 95%). 1H NMR (300 MHz, CDCl3)
d 8.57 (1H, m, CHAr), 7.68 (1H, m, CHAr), 7.22–7.13 (2H, m, CHAr),
4.56 (2H, s, CH2), 4.10 (2H, m, CH2), 2.22 (1H, t, J4 2.4 Hz, „CH),
1.49 (9H, m, CH3); 13C NMR (75 MHz, CDCl3) d 156.3 (CO), 155.1
(CAr), 149.3, 136.6, 122.1, 120.9 (CHAr), 80.7 (CCH), 79.3 (CtBu),
79.2 („CH), 57.7 (NCH2), 36.5 (NCH2), 28.3 (CH3); MS (DCI/NH3):
247 [M+H+]+.
Preparative reversed-phase HPLC was accomplished on
a
Waters PREP LC 4000 chromatography system with a (DEDL) PL-
ELS 1000 photodiode array detector or a Waters 600E gradient
chromatograph coupled to a UV–visible detector (ICS) and a
c-
detector (Raytest) for the 99mTc-compounds. Non-radioactive HPLC
samples were purified on a preparative Prevail C-18 column
(2.2 ꢁ 25 cm) for 10, an NH2 Alltech Altima 5
lM C-18 column
for 6, 7, 8, 6-Re, 8-Re and a Phenomenex Luna C-18 column for
7-Re. Gradient A: the mobile phase was H2O (solvent A) and CH3CN
(solvent B). The gradient consisted of 100% A for 5 min then 100%
A–100% B in 35 min (20 mL/min flow rate). Gradient B: the mobile
phase was HCOONH4 buffer, 10 mM, pH 4 (solvent A) and CH3CN
(solvent B). The gradient consisted of 90% A–50% A in 18 min then
50% A–90% A in 2 min (20 mL/min flow rate).
RP-HPLC analysis and purification of 99mTc-complexes were
carried out on a Nucleodur C18 end capped RP column (Mache-
rey-Nagel analytical column 125 ꢁ 4 mm, 5
lm). The RP-HPLC
conditions were as follows: flow rate was 1 mL/min, eluent 1
(for complexes 6-Tc, 7-Tc and 10-Tc) was 0.1% TFA in H2O/MeOH
80:20 (solvent A) and 0.1% TFA in H2O/MeOH 40:60 (solvent B)
and gradient system was 0–10 min, 100% A–100% B; 10–
15 min, 100% B; 15–20 min, 100% B–100% A, eluent 2 (for com-
plexes 8-Tc) was 0.1% TFA in H2O/MeOH 52:48 and isocratic gra-
dient. In all runs, the wavelength used for UV detection was
230 nm.
3.5. N-Propargyl-2-picolylamine 4
N-Boc,N-propargyl-2-picolylamine (4.75 g, 19.3 mmol) was
dissolved in CH2Cl2–TFA (1:1; 15 mL) and stirred at room tem-
perature for 3H, then the solvent was removed. The free amine
was obtained by dissolving the residue in 0.5 M NaOH (10 mL)
and extracting into CHCl3 (3 ꢁ 30 mL). The organic phases were
collected, dried over Na2SO4 and evaporated under reduced pres-
sure. The crude product was purified by chromatography on
silica gel (CH2Cl2–MeOH 95:5) to give 4 as a pale yellow oil
(2.67 g, 95%). Analytical data were in agreement with the
literature.17
Caution: Sodium azide, when inhaled, is highly toxic. Precau-
tions must be taken when weighing the material, such as using a
powder mask and a teflon spatula (metallic spatula may cause
explosion). The azidation reactions should be performed behind a
plastic shield due to the potential explosion. In acidic pH hydrazoic
acid (HN3) may be formed, which may explode and/or, when in-
haled, may cause intoxication.
3.6. General procedure for the synthesis of Re(CO)3 complexes
Complexes 1-Re and 2-Re were prepared by a substitution route
from commercial Re(CO)5Br. Ligand 1 or 2 (1 mmol) and Re(CO)5Br
(406 mg, 1 mmol) were dissolved in MeOH (10 mL) and stirred at
65 °C for 6 h. After cooling to room temperature, the brown solution
was evaporated to dryness. The residue was purified by column
chromatography on silica gel (CH2Cl2–MeOH 95:5) to give the
desired complex of general formula [Re(CO)3L][Br].
3.2. 3-[Bis(2-quinolinylmethyl)amino]-prop-1-yne 2
2-Chloromethylquinoline monochloride (1.5 g, 7 mmol) and
potassium carbonate (4.83 g, 35 mmol) were added to acetonitrile
(20 mL) and stirred at room temperature for 15 min. Propargyl
amine (0.24 mL, 3.5 mmol) was added and the mixture stirred at
75 °C overnight. The solvent was evaporated and the residue puri-
fied by column chromatography on silica gel (CH2Cl2–MeOH 98:2).
The product was isolated as a brown powder (730 mg, 62%). 1H
NMR (300 MHz, CDCl3) d 8.10 (4H, m, CHAr), 7.50 (6H, m, CHAr),
7.24 (2H, m, CHAr), 4.08 (4H, s, CH2), 3.46 (2H, m, NCH2), 2.33
(1H, t, J4 2.3 Hz, „CH); 13C NMR (75 MHz, CDCl3) d 159.5, 147.1,
127.6 (CAr), 136.5, 129.5, 129.4, 127.5, 126.2, 122.0 (CHAr), 78.7
(CAr), 73.7 („CH), 60.3 (NCH2), 38.0 (NCH2); MS (DCI/NH3): 338
[M+H+]+.
3.7. Complex 1-Re
237 mg of 1 gave 1-Re as a brown powder (430 mg, 74%). 1H
NMR (300 MHz, CDCl3) d 8.64 (2H, m, CHAr), 7.92 (2H, m, CHAr),
7.82 (2H, m, CHAr), 7.20 (2H, m, CHpyr), 5.97 (2H, d, J 18 Hz,
CH2), 4.66 (2H, d, J 18 Hz, CH2), 4.48 (2H, m, NCH2), 2.78 (1H, t,
J4 2.5 Hz, „CH); 13C NMR (75 MHz, CDCl3) d 195.4 (C„O),
161.6 (CAr), 151.0, 140.4, 125.9, 125.5 (CHAr), 80.2 (CCH), 76.1
(„CH), 67.7 (NCH2), 58.2 (NCH2); MS (ES+): 506/508
[(1-Re)ꢀBrꢀ]+.
3.3. N-Boc-2-picolylamine
According to a modification of a literature procedure,20 to a
solution of di-tert-butyl dicarbonate (21.88 g, 0.1 mol) in CH2Cl2
(200 mL) at 0 °C, 2-(aminomethyl)pyridine (10.30 mL, 0.1 mol)
was added dropwise. The resulting solution was stirred at room
temperature overnight, and then diluted with 100 mL of CH2Cl2.
The organic layer was washed twice with 0.1 M NaOH (100 mL),
dried over Na2SO4 and evaporated to give a pale yellow oil
(20.70 g, 99%). 1H NMR (300 MHz, CDCl3) d 8.48 (1H, m, CHpyr),
7.70 (1H, m, CHAr), 7.31–7.17 (2H, m, CHAr), 4.37 (2H, d, J 5.5 Hz,
CH2), 1.40 (9H, s, CH3); MS (DCI/NH3): 209 [M+H+]+.
3.8. Complex 2-Re
337 mg of 2 give 2-Re as a brown powder (300 mg, 41%). 1H
NMR (300 MHz, CDCl3) d 8.51 (2H, m, CHAr), 8.38 (2H, m, CHAr),
7.93–7.83 (6H, m, CHAr), 7.68 (2H, m, CHAr), 6.10 (2H, d, J
17.9 Hz, CH2), 5.33 (2H, d, J 17.9 Hz, CH2), 4.76 (2H, m, NCH2),
2.72 (1H, t, J4 2.5 Hz, „CH); 13C NMR (75 MHz, MeOD) d 195.7,
193.6 (C„O), 164.7, 146.8, 128.5 (CAr), 141.4, 132.7, 128.5, 128.1,
127.8, 119.5 (CHAr), 79.3 (CCH), 76.4 („CH), 68.8 (NCH2), 56.0
(NCH2); MS (FAB+): 606/608 [MꢀBrꢀ]+.

32
E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
3.9. 2,3,4,6-Penta-O-acetyl-b-
D
-glucopyranosyl azide 5
(2H, s, CHAr), 5.67 (1H, d, J1,2 9.2 Hz, H-1), 5.40 (3H, br, OH), 5.10
(2H, t, CHHAr), 5.02 (2H, m, CH2Trz), 4.64 (3H, dd, 2 ꢁ CHHAr + OH),
4.58 (1H, d, J 17 Hz, CHHAr), 3.77 (2H, m, H-2, H-6), 3.49 (3H, m, H-
3, H-4, H-60), 3.49 (2H, m, H-5, H-60); 13C NMR (125 MHz, DMSO-
d6): d 196.1, 195.2 (CO), 160.3 (CAr), 151.8 (CAr), 140.5 (CAr), 139.4
(CAr), 125.9, 125.7 (CHTrz, CAr), 123.5 (CAr), 87.7 (C-1), 80.0, 76.7
(C-3, C-4), 72.4 (C-2), 69.6 (C-5), 67.5, 67.4 (CH2Ar), 62.8, 60.7
(CH2); HRMS (ES+): found 711.1356 C24H26N6O8Re requires
711.1342 [MꢀBrꢀ]+.
1,2,3,4,6-Penta-O-acetyl-b-
was dissolved in dry DCM (200 mL) at rt. TMSN3 (5.25 mL,
39 mmol) and stannic chloride (900 L, 7.6 mmol) were added
D
-glucopyranoside (6 g, 15 mmol)
l
dropwise to the solution and the mixture was stirred for 15 min
at room temperature under Ar. NaHCO3 (7.68 g, 90 mmol) was
added and the mixture diluted with CH2Cl2 (200 mL) prior to
extraction with sat NaHCO3 (300 mL) and water (300 mL). The or-
ganic layer was dried over Na2SO4, filtered and evaporated under
reduced pressure. The residue was purified by flash chromatogra-
phy on silica gel (6:4 cyclohexane–EtOAc) to give pure 2,3,4,6-pen-
3.14. General procedure for the Click chemistry with CuSO4–
NaAsc
ta-O-acetyl-b-D
-glucopyranosyl azide (4 g, 70% yield).32
This compound (1 g, 2.68 mmol) was dissolved in technical
grade MeOH (50 mL) and one molar sodium methanolate solution
(1 mL) was added. The mixture was stirred for 8 h and 120H+ resin
was added until the pH reached 5. The resin was filtered and the
filtrate evaporated under reduced pressure to give pure glucopyr-
anosyl azide 5 (542 mg, 99% yield) without purification. Analytical
data were in agreement with the literature.21
Compounds 8, 8-Re and 9 were synthesized using this proce-
dure. Alkynyl compound 2, 2-Re or 3 (73
syl azide 5 (73 mol) were dissolved in a dioxane–H2O mixture
(5:1; 1.2 mL). CuSO4 (14 mol) and sodium ascorbate (30 mol)
lmol) and glucopyrano-
l
l
l
were added and the mixture was stirred at 100 °C for 30 min under
microwave irradiation. The mixture was filtered on a pad of Celite
and rinsed with MeOH. The filtrate was evaporated under reduced
pressure to give the expected cycloadduct.
3.10. General procedure for the heterogeneous Click chemistry
3.15. Glycoconjugate 8
Compounds 6, 7, 6-Re were obtained accordingly. The catalyst
was synthesized by immobilisation of copper nanoparticles onto
charcoal as previously described.20 Alkynyl compound 1, 4 or 1-
½
a 2D0
ꢂ
ꢀ10 (c 0.1, H2O); Rt = 36.66 min (gradient A); 1H NMR
(300 MHz, CD3OD) d 8.27 (1H, s, CHTrz), 8.18 (2H, d, J 8.5 Hz, CHAr),
7.92 (2H, d, CHAr), 7.78 (2H, d, J 8.4 Hz, CHAr) 7.75 (2H, d, J 8.4 Hz,
CHAr), 7.65 (2H, t, CHAr), 7.48 (2H, t, J 7.0 Hz, CHAr), 5.64 (1H, d, J1,2
9.2 Hz, H-1), 3.98–3.87 (8H, m, H-2, H-6, 2 ꢁ CH2Ar, CH2Trz), 3.72
Re (146 lmol) and glucopyranosyl azide 5 (146 lmol) were dis-
solved in a mixture of t-BuOH and H2O (5:1; 1.2 mL). Catalyst C/
Cu (35 mg) was added and the mixture was stirred at 100 °C under
microwave irradiation until TLC or ESI-MS indicated total disap-
pearance of the starting materials. The mixture was filtered over
a pad of Celite and rinsed with methanol. The filtrate was evapo-
rated under reduced pressure to give the expected cycloadducts.
(1H, dd, J6,6 11 Hz, J5,6 3 Hz, H-60), 3.62–3.50 (3H, m, H-3,4,5);
13C NMR (75 MHz, CD3OD): d 161.2 (CAr), 148.9 (CAr), 145.1
(NC@CHAr), 138.5, 130.9, 128.9, 128.8, 127.6 (4CHAr, CAr), 124.7
(NC@CHTrz), 122.5 (CHAr), 89.6 (C-1), 81.1, 78.5, 74.1, 70.9 (C-2, 3,
4, 5), 62.3 (C-6), 61.1 (2CH2Ar), 50.2 (CH2Trz); HRMS (ES+): found
565.2151 C29H30N6O5Na requires 565.2175 [M+Na+]+.
0
0
3.11. Glucoconjugate 6
½
a 2D0
ꢂ
ꢀ7 (c 0.1, MeOH); Rt = 23.68 min (gradient A); 1H NMR
3.16. Glycocomplex 8-Re
(500 MHz, DMSO) d 8.48 (2H, d, J 4.0 Hz, CHAr), 8.27 (1H, s, CHTrz),
7.76 (2H, dd, J 7.6 Hz, J 1.6 Hz, CHAr), 7.58 (2H, d, J 7.8 Hz, CHAr),
7.25 (2H, dd, CHAr), 5.52 (1H, d, J1,2 9.2 Hz, H-1), 5.36 (1H, d, J
5.8 Hz, OH), 5.26 (1H, br, OH), 5.13 (1H, d, OH), 4.63 (1H, t, J
4.8 Hz, OH), 3.70 (8H, m, 2 ꢁ CH2, CH2Trz, H-2, H-6), 3.44 (2H, m,
H-3, H-60), 3.50 (1H, H-4), 3.25 (1H, br, H-5); 13C NMR (125 MHz,
DMSO-d6): d 159.0 (CAr), 148.9 (CAr), 143.4 (CqTrz), 136.6 (CAr),
122.9 (CHTrz), 122.6, 122.1 (CAr), 87.5 (C-1), 80.0, 77.0 (C-3, C-4),
72.1 (C-2), 69.6 (C-5), 60.7 (C-6), 58.7 (CH2), 48.1 (CH2); HRMS
(ES+): found 465.1861 C21H26N6O5Na requires 465.1862 [M+Na+]+.
½
a 2D0
ꢂ
ꢀ13 (c 0.1, MeOH); Rt = 21.02 min (gradient A); 1H NMR
(300 MHz, CD3OD) d 8.68 (1H, s, CHTrz), 8.52 (4H, br, CHAr), 8.00
(3H, m, CHAr), 7.87 (1H, t, J 8 Hz, CHAr), 7.66 (2H, t, J 8 Hz, CHAr),
7.60 (2H, dd, J 8 Hz, J 5 Hz, CHAr), 5.77 (1H, d, J1,2 9 Hz, H-1), 5.45
(1H, m, CHH), 5.24 (2H, s, CH2Trz), 4.90 (3H, m, 3 ꢁ CHHAr), 4.03
(2H, m, H-2, H-6), 3.70 (1H, dd, J6,6 11 Hz, J5,6 3 Hz, H-60), 3.65–
3.55 (3H, m, H-3, H-4, H-5); 13C NMR (75 MHz, CD3OD): d 197.2,
195.2 (CO), 166.0, 148.1 (C), 142.9 (CHAr), 141.2 (NC@CHTrz),
133.6, 133.5, 130.9, 129.8, 129.5 (CHAr), 127.5 (NC@CHTrz), 121.3
(CHAr), 89.7 (C-1), 81.2, 78.1 (C-3, C-4), 74.2 (C-2), 70.7 (C-5),
69.6, 62.3, 62.1 (CH2); HRMS (ES+): found 811.1647 C32H30N6O8Re
requires 811.1655 [MꢀBrꢀ]+.
0
0
3.12. Glycoconjugate 7
½
a 2D0
ꢂ
ꢀ11 (c 0.1, MeOH); Rt = 26.73 min (gradient A); 1H NMR
(300 MHz, CD3OD) d 8.49 (1H, br, CHAr), 8.13 (1H, s, CHTrz), 7.80
(1H, t, J 7.5 Hz, CHAr), 7.47 (1H, d, J 6.9 Hz, CHAr), 7.30 (1H, br, CHAr),
5.62 (1H, d, J1,2 9.1 Hz, H-1), 3.91–3.85 (6H, m, H-2, H-6, 2CH2),
3.72 (1H, dd, H-60), 3.60–3.50 (3H, m, H-3, H-4, H-5); 13C NMR
(75 MHz, CD3OD): d 159.9 (CAr), 149.9 (CHAr), 146.9 (NC@CHTrz),
138.8, 124.2 (CHAr), 123.8 (NC@CHTrz), 123.6 (CHAr), 89.6 (C-1),
81.1, 78.4, 74.0, 70.9 (C-2, 3, 4, 5), 62.4 (C-6), 54.4, 44.3
(2xCH2NH2); HRMS (ES+): found 374.1428 C15H21N5O5Na requires
374.1440 [M+Na+]+.
3.17. Glycoconjugate 9
½
a 2D0
ꢂ
ꢀ6 (c 0.3, CH2Cl2); 1H NMR (300 MHz, CD3OD) d 8.12 (1H,
br, CHTrz), 5.61 (1H, d, J1,2 9.2 Hz, H-1), 4.03–3.30 (12H, m, H-2glu to
H-6glu, 3 ꢁ CH2), 1.45 (18H, s, 6 ꢁ CH3); 13C NMR (75 MHz, CD3OD):
d 171.8 (CO), 145.1 (NC@CHTrz), 124.6 (NC@CHTrz), 89.5 (C-1), 82.4,
78.3 (C-3, C-4), 74.0 (C-2), 70.8 (C-5), 62.3 (C-6), 55.7
(CH2Ar+CHTrz), 28.4 (CH3); HRMS (ES+): found 511.2364
C21H36N4O9Na requires 511.2380 [M+Na+]+.
3.13. Glycocomplex 6-Re
3.18. Glycoconjugate 10
½
a 2D0
ꢂ
ꢀ9 (c 0.1, MeOH); Rt = 22.28 min (gradient A); 1H NMR
Compound 9 (300 mg, 0.615 mmol) was dissolved in ethanol
(12 mL). 2 M NaOH (12 mL) was added while stirring, and the mix-
ture was heated at 60 °C for 12 h. After evaporation under reduced
(500 MHz, DMSO) d 8.82 (2H, s, CHAr), 8.63 (1H, s, CHTrz), 7.97
(2H, dd, J 7.8 Hz, J <1 Hz, CHAr), 7.55 (2H, d, J 7.8 Hz, CHAr), 7.40

E. Benoist et al. / Carbohydrate Research 346 (2011) 26–34
33
pressure, the residue was dissolved in water (12 mL) at 0 °C. 6 M
3.22. Biodistribution in healthy rats
HCl was added until the solution became acidic. The mixture was
evaporated under reduced pressure and the dry residue dissolved
in absolute ethanol (12 mL). After salts were filtered on a frit, the
filtrate was evaporated under reduced pressure leading to 10
All experiments were carried out in compliance with French
laws relating to the conduct of animal experimentation. Before
being used in the animal studies, purified 99mTc-complex solution
quantitatively as a hygroscopic solid. ½a D20
ꢂ
ꢀ5 (c 0.1, H2O);
was filtered through a 0.22 l
m sterile filter (MilliporeÒ) and di-
Rt = 4.54 min (gradient A); 1H NMR (300 MHz, DMSO-d6) d 8.23
(1H, s, CHTrz), 5.52 (1H, d, J1,2 8.6 Hz, H-1), 4.08 (2H, br, CH2),
3.72–3.33 (9H, m, H-2,3,5,6,60, 2 ꢁ CH2), 3.23 (1H, br, H-4); 13C
NMR (75 MHz, DMSO-d6): d 171.0 (CO), 141.9 (NC@CHTrz), 123.8
(NC@CHTrz), 87.5 (C-1), 80.0, 76.8 (C-3, C-4), 72.2 (C-2), 69.5 (C-
5), 60.7 (C-6), 53.4 (CH2CO2H), 48.2 (CH2Trz); HRMS (ES+): found
399.1121 C13H20N4O9Na requires 399.1128 [M+Na+]+.
luted with sterile saline solutions. Healthy male Wistar rats
(200–250 g) anaesthetised with NesdonalÒ were sacrificed at 5,
30 and 240 min post-injection (p.i.) (n = 3) after an intra-jugular
injection of 300 lL of the diluted tracer solution 6-Tc, 7-Tc or 10-
Tc. The organs of interest (liver, spleen, heart, lungs, kidneys and
brain) were dissected, weighed and their radioactivity was mea-
sured in a Packard autogamma counter. Results, expressed as per-
centage of injected dose per organ (%ID/organ) were summarized
in Table 2. For total blood radioactivity calculation, blood mass is
assumed to be 7% of total body mass.
3.19. Glycocomplex 7-Re
Glycoconjugate
7
(100 mg, 0.284 mmol) and Re(CO)5Cl
(113 mg, 0.312 mmol) were dissolved in MeOH (10 mL) and stir-
red at 65 °C for 6 h. After cooling to room temperature, the solu-
tion was evaporated to dryness and purified by preparative C-18
HPLC to give the desired complex 7-Re as a white powder.
(140 mg, 75% yield); Rt = 5.79/6.03 min (gradient B); 1H NMR
(300 MHz, DMSO-d6) d 8.75 (1H, m, CHAr), 8.51 (1H, m, CHTrz),
8.02 (1H, t, J 7.5 Hz, CHAr), 7.66 (1H, d, J 7.0 Hz, CHAr), 7.42 (1H,
br, CHAr), 5.61 (1H, d, J1,2 8.7 Hz, H-1), 4.76 (2H, s, CH2), 4.54
(1H, m, CHH), 4.36 (1H, m, CHH), 3.65–3.22 (6H, m, H-3, H-4, H-
5, H-6, H-60); 13C NMR (75 MHz, DMSO-d6): d 196.5–195.5 (CO),
161.4 (CAr), 153.4 (CHAr), 142.6 (NC@CHAr), 141.4, 127.3 125.7
(CHAr), 123.2 (NC@CHTrz), 89.2 (C-1), 81.4, 80.1, 77.1, 73.2 (C-2,
3, 4, 5), 62.9 (CH2N), 60.9 (C-6), 51.8 (CH2N). All the peaks are dou-
bled due to the presence of the two isomers. MS (ES+): 622.3
[MꢀClꢀ]+. Anal. Calcd for C18H21N5O8ReCl: C, 32.90; H, 3.22.
Found: C, 32.34; H, 3.27.
Acknowledgements
This work was carried out with financial support from the
French Agence Nationale de la Recherce (ANR JC07_183019), the
Centre National de la Recherche Scientifique, the Ministère
Délégué à l’Enseignement Supérieur et à la Recherche and the
Conseil Régional de Picardie.
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3.20. Radiolabelling of glycoconjugates
Na[99mTcO4] was eluted from a 99Mo/99mTc generator (Mallinck-
rodt Inc.) using 0.9% saline solution. The precursor [99mTc(CO)3(-
H2O)3]+ was prepared from pertechnetate (600
lL eluate) using
an Isolink kitÒ.33 The mixture was stirred for 30 min at 95 °C and
then cooled down.
To a solution of the glycoconjugate 6, 7, 8 or 10 (1 mg/mL in
methanol, 100
were added successively 200
0.2 M pH 3.4 and 65
L of the freshly prepared [99mTc(CO)3
lL) placed in a borosilicate vial under nitrogen,
l
L of an aqueous acetic acid buffer
l
(H2O)3]+ solution. The vial was sealed with a teflon-lined cap
and the mixture was heated at 95 °C for 30 min. Upon cooling,
the resulting complex was analysed and purified with the HPLC
system described above. The radiolabelling yields ranged from
95% for 6-Tc and 7-Tc to 90% for 8-Tc and 80% for 10-Tc. The
radiochemical purity assessed by ITLC was >95% after RP-HPLC
purification for each complex. The retention time of 6-Tc, 7-Tc,
8-Tc and 10-Tc were 6.44 min, 5.08/5.60 min, 8.13 min and
4.69 min, respectively.
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by RP-HPLC.

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