Lanthanide(III) complexes of pyridine-tetraacetic acid-glycoconjugates: Synthesis and luminescence studies of mono and divalent derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.03.003

A potent lanthanide chelate, fulfilling the requirements for the development of MRI contrast agents or luminescent probes, was armed with alkyne groups. We then implemented a click methodology to graft the bifunctional ligand to azide-containing glucoside and maltoside scaffolds. The resulting hydrophilic glycoconjugates retained the ligand binding capacity for Eu ³⁺ or Tb³⁺ ion as evidenced by the number of bound water molecules to the lanthanide ion. Divalent Eu³⁺ and Tb³⁺ complexes were shown to double the brightness of the emitted fluorescent signal compared to its monovalent derivatives. Designing multivalent lanthanide luminescent probes would enable the fluorescent signal of labeled biomolecules to be enhanced.

Full text of DOI:10.1016/j.bmcl.2012.03.003

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2684–2688
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lanthanide(III) complexes of pyridine–tetraacetic acid-glycoconjugates:
Synthesis and luminescence studies of mono and divalent derivatives
a,b,
⇑
a
c,d
a
a
Sébastien G. Gouin
, Mélissa Roger , Nadine Leygue , David Deniaud , Karine Julienne ,
c,d
c,d
b
c,d,
⇑
Eric Benoist , Claude Picard , José Kovensky , Chantal Galaup
LUNAM Université, CEISAM, Chimie Et Interdisciplinarité, Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR des Sciences et des Techniques, 2, rue de la Houssinière, BP
2208, 44322 NANTES Cedex 3, France
Laboratoire des Glucides FRE 3517, Institut de Chimie de Picardie, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex 1, France
CNRS, Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique, SPCMIB, UMR CNRS-5068, 118 route de Narbonne, F-31062 Toulouse Cedex 04, France
Université Paul Sabatier, UPS, Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique, SPCMIB, UMR CNRS-5068, 118 route de Narbonne, F-31062 Toulouse Cedex
a
9
b
c
d
04, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A potent lanthanide chelate, fulfilling the requirements for the development of MRI contrast agents or
luminescent probes, was armed with alkyne groups. We then implemented a click methodology to graft
the bifunctional ligand to azide-containing glucoside and maltoside scaffolds. The resulting hydrophilic
Received 23 January 2012
Revised 2 March 2012
Accepted 3 March 2012
Available online 8 March 2012
3+
3+
glycoconjugates retained the ligand binding capacity for Eu or Tb ion as evidenced by the number
3
+
3+
of bound water molecules to the lanthanide ion. Divalent Eu and Tb complexes were shown to double
the brightness of the emitted fluorescent signal compared to its monovalent derivatives. Designing multi-
valent lanthanide luminescent probes would enable the fluorescent signal of labeled biomolecules to be
enhanced.
Keywords:
Glycoconjugates
Chelating agents
Lanthanides
Ó 2012 Elsevier Ltd. All rights reserved.
Luminescence
Click chemistry
The development of metal–carbohydrate conjugates for cellular
imaging and clinical diagnosis is an extensive field of research. A
wide range of glycoconjugates bearing contrast agents and radioel-
ements, where the sugar moieties serve as biocompatible scaffolds
or ligands to target specific organs, has been described. In nuclear
medicine, several research groups have explored the development
of glucose conjugates as new radiotracers in tumor oncology. Ap-
proaches generally consist of grafting stable radioactive techne-
photophysical properties of lanthanide probes are particularly sui-
ted for the time-gated luminescence detection of biological events.
8
3
+
3+
3+
They combine narrow bands in the visible region (Eu , Tb , Sm ,
3
+
3+
3+
Dy ) and NIR region (Nd , Yb ), long excited-state lifetimes (ls
9
and ms range) and large Stokes’s shifts. The emission of lanthanide
ions is usually carried out by an indirect process called the ‘antenna
effect’ in which energy from the excited state of a sensitizer (a chro-
mophoric group incorporated into a chelating unit) transfers to the
excited states of the lanthanide ion, which emits in its emissive re-
gion. The chelating unit is expected to form water-soluble lantha-
nide complexes of high kinetic stability and to provide an efficient
shielding of the Ln ion to prevent unwanted non-radiative deacti-
vations from surrounding water molecules. In the course of re-
search on bimodal lanthanide probes suitable for optical and
magnetic resonance imaging, we and others have shown that
Ln complexes derived from pyridine–tetraacetic acid possess
the physical requirements for MRI (Ln = Gd) and optical imaging
(Ln = Eu, Nd).
9
9m
tium (Tc
) chelates to glycoside scaffolds with the aim of
1
0
selectively targeting and irradiating cancer cells overexpressing
glucose transporters (GLUTs).1 Gadolinium complexes are com-
monly used in magnetic resonance imaging (MRI) to enhance local
contrast. Starch and inulin polysaccharides bearing multiple Gd(III)
were shown to exhibit both excellent biocompatibility and high
,2
3
+
1
1
3,4
12
13
molecular relaxivity leading to enhanced signals. Interestingly,
much shorter saccharide scaffolds, such as b-cyclodextrin and tria-
minoglycoside chitosan, functionalized with seven and three Gd-
DTPA, respectively, were shown to enhance the relative signal
3
+
5
,6
intensity per Gd.
In this context, we describe herein a synthetic method to tether
a new functionalized pyridine–tetraacetic ligand to glucoside and
maltoside scaffolds (Fig. 1). We wanted to assess if the complexing
abilities and the photophysical properties of such chelates were re-
tained after their tethering to the sugar moieties. The alkynyl-
armed pyridine chelate with pendant iminodiacetic groups was
Luminescent carbohydrate-based lanthanide probes for sensing
7
biological substrates have been much less explored. The
⇑
Corresponding authors.
E-mail address: sebastien.gouin@univ-nantes.fr (S.G. Gouin).
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.003

S. G. Gouin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2684–2688
2685
COO-
COO-
N
COO-
COO-
COO-
COO-
O
N Ln3+
N
COO-
COO-
NH
O
NH
N
N Ln3+
N
N
N
1.Ln
N
OH
Two grafting
points
N
O
O
HO
HO
N
HO
HO
N
OH
HO
2.Ln
HO
Two Valencies
COO-
COO-
N
O
N Ln3+
COO-
COO-
NH
N
N
N
O
N
N
H
N
COO-
O
HO
HO
N
N
COO-
Ln3+
HO
N
N
N
O
COO-
HO
COO-
O
HO
OH
3.Ln
Figure 1. Structure of the lanthanide(III) complexes of the functionalized pyridine–tetraacetic acid glycoconjugates 1, 2 and 3.
grafted by click chemistry to hydrophilic and biocompatible gluco-
side and maltoside scaffolds. The bifunctional pyridine-based li-
gand was grafted onto either the C-6 or the anomeric position of
the sugars (compounds 1 and 2). In fact, the specific topologies
adopted by the scaffold may prevent the access of water molecules
to the inner-sphere coordination site, as previously described for
1
4
Gd complexes. The grafting position may therefore be detrimen-
tal to the efficiency of the probes. The divalent compound 3 was
designed to accommodate two lanthanide ions. The modulation
of the valency in 1 and 3 does not introduce new functional groups
and should not significantly affect the hydrophilicity and spatial
presentation of the pyridine-based chelate, because a critical glu-
coside fragment is repeated. The presence of several chelating
agents tethered to a common scaffold is useful for delivering a lar-
Figure 2. Structure of the azido-functionalized carbohydrates 4–6.
ger payload of lanthanides to a specific target or for enhancing sig-
nal intensity with Gd ions.3
–6,15
We have recently reported the
We then focused our effort on the synthesis of the pyridine–tet-
raacetic acid ligand bearing an alkynyl group (Scheme 1). Scharbert
and co-workers previously described the radical alkylation of
synthesis of a set of bifunctional carbohydrate cores with up to se-
ven azides and bearing a terminal amino group.16 These scaffolds
may serve as valuable platforms for the delivery of lanthanide che-
lates to specific biological receptors. The chemical strategy devel-
oped here and the analysis of the photophysical properties of the
new lanthanide–carbohydrate conjugates give first insights into
the possibility of designing multivalent luminescent lanthanide
chelates.
We first designed azido-functionalized carbohydrate scaffolds
–6 (Fig. 2). Compounds 5 and 6 were directly obtained from
unprotected glucose and maltose, respectively, by a protocol that
we previously implemented for the selective azidation of carbohy-
20
methyl isonicotinate with ammonium persulfate and silver salts
with 30% yields. In our laboratory, these conditions led to com-
pound 7 with less than 10% yield. We were delighted to see that
a significantly higher yield of 60% was reached when the silver salt
was substituted with iron dichloride. Phosphorus tribromide
allowed the conversion of the diol 8 to 9 with 71% yield. The two
bromide ions were successfully substituted by di-tert-butylimino-
diacetate in acetonitrile to provide 10. Selective hydrolysis of the
417
2
1
methyl ester group led to the corresponding carboxylic acid 11
quantitatively (overall yield: 40%). In a similar manner, Lebeau22
obtained 11 in 4 steps starting from methyl isonicotinate; how-
ever, no characterization was indicated. After in situ activation of
this group by PyBOP, and coupling with 3-butynyl amine,23 the
1
8
drates. The ‘one-pot’ method consists of adding a PPh
3
:CBr
mixture in a 2:2:10 ratio relative to the sugar unit, followed
by acetylation of the crude mixture.
4
:
1
9
NaN
3

2
686
S. G. Gouin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2684–2688
O
OMe
O
OMe
O
OMe
a
b
N
N
N
OH
OH
Br
Br
7
8
9
c
H
N
O
OX
O
ButO C
CO2tBu
2
ButO C
CO2tBu
2
e
ButO2C
N
d
N
CO tBu
2
ButO C
N
N
CO tBu
N
2
2
N
1
1
0 x = Me
1 X = H
1
2
Scheme 1. Synthesis of the alkynyl-armed bifunctional chelate 12. Reagents and conditions: (a) (NH)
0 °C, 17 h, 60%; (b) PBr (3 equiv), CH CN, 60 °C, 8 h, 71%; (c) di-tert-butyl iminodiacetate (2 equiv), Na
reflux, 2 h then HCl, quant.; (e) 3-butyn-1amine hydrochloride (1.3 equiv), DIPEA (5 equiv), PyBOP (1.6 equiv) CH
4
S
2
O
CO
8
(10 equiv), FeCl
2
Á4H
2
O (0.25 equiv), H
2
SO
4
cat., MeOH/H
2
O (1/1),
O (2/1),
5
3
3
2
3
, CH
3
CN, 100 °C, 16 h, 95%; (d) K
Cl , rt, 24 h, 62%.
2
CO
3
(1 equiv), MeOH/H
2
2
2
bifunctional chelate 1221 was obtained with 62% yield, after chro-
matographic purification on alumina.
at 276 nm; this absorption band is typical of the
p
–p
⁄
transitions
attributed to the pyridine moiety. The values of the absorption
We then developed a method to tether 12 to the azido-function-
alized carbohydrate cores 4, 5 and 6 with a single purification step
coefficients of the complexed glycoconjugates 1, 2 and 3 (
e
ꢀ2500
À1
À1
vs 5600 M cm ) are in agreement with the presence of one or
two chelating agents grafted onto the carbohydrate scaffolds. Upon
excitation in this absorption band, both europium and terbium
complexes display sizeable and structured lanthanide emission
luminescence in the visible spectra region (Fig. 3) characteristic
of the lanthanide ion, indicating that the energy is absorbed by
the chromophoric group and efficiently transferred to the chelated
(
Scheme 2). A copper-catalyzed azide alkyne cyclization (CuAAC)
2
4
protocol was first performed in a mixture of dioxane and water.
We recently successfully applied this click protocol to the design of
radiolabeled glucose conjugates.2f Stoichiometric (and not cata-
lytic) amounts of copper sulfate were used for the cyclization,
due to the high potency of the compounds to chelate and inactivate
copper. The formation of such complexes was shown by ESI-MS
analysis of the crude mixture. Extraction with an EDTA solution
provided the protected glycoconjugates free of copper by a trans-
chelation mechanism. The exclusive formation of the 1,4-regioi-
3
+
5
7
3+
0 2
Ln ion. The most intense peaks are the transitions D ? F (Eu )
5
7
3+
and D
The luminescence lifetimes (
measured in aerated Tris buffer solutions at pH 7.4. As presented
in Table 1, the luminescence properties ( ) of the pyridine-gly-
coconjugates are quite similar within the europium complexes
1.Eu, 2.Eu and 3.Eu ( = 0.39 ms, 0.4–0.6%) and the terbium com-
plexes 1.Tb, 2.Tb and 3.Tb ( = 1.18 ms, 2.3–3%). The comparison
of the dependence of the lifetimes of the complexes in H O (
and D O ( ) at 298 K allows an assessment of the hydration state
of the Ln using Parker’s equation.
4
? F
5
(Tb ) at 616 and 545 nm, respectively.
s
) and quantum yields (
U) were
somers was confirmed by the large
D
(dC-4–dC-5) values (>20 ppm)
s, U
1
3
25
observed by C NMR. Protecting groups were sequentially re-
moved using an aqueous solution of ammonia for the acetates
and, after evaporation, by dissolution in pure TFA for the tert-butyl
groups. Crude glycoconjugates were finally purified by preparative
s
U
s
U
2
H
s )
26
HPLC on a C-18 column to lead to pure 1 , 2 and 3.
2
s
D
3
+
3+
27
The Ln complexes (Ln = Eu, Tb) of the glycoconjugates 1 and 2
were readily prepared by mixing equimolar quantities of the lan-
thanide salt (LnCl , 6H O) in water. For glycoconjugate 3, two
3 2
The value of the hydration number q of 2 is in agreement with
the seven complexation sites of the grafted chelate, and matches
the literature value for the corresponding unfunctionalized pyri-
dine chelate. Additionally, the grafting position of the chelate
to the sugar does not significantly modulate the luminescence
properties and the sugar scaffolds do not seem to interfere with
equivalents of lanthanide salt were used. The solutions were then
À5
12
adjusted to a final concentration of 1 Â 10 M in Tris buffer (pH
7
.4, 50 mM). The UV–vis absorption spectra of the Eu and Tb
complexes display broad bands in the UV domain with maxima
R2
R4
O
O
3
R = OH, R4 = R 43%
5
1
AcO
HO
HO
R3
AcO
R1
3
5
4
2 R = R , R = OH 52%
AcO
HO
1
2
i) 12, CuSO4,
NaAsc,
4
5
R = OAc,R = N3
R = N R2 = OAc
N3
1
R5
3
,
dioxane-H O
2
O
ii) NH -MeOH
AcO
AcO
3
O
R5
N3
HO
HO
iii) TFA
AcO
HO
O
O
O
AcO
O
OAc
N
3 20%a
HO
OH
6
AcO
HO
COOH
N
N
N
N
COOH
NH
N
O
R5
COOH
COOH
a
Scheme 2. Synthetic procedure to obtain glycoconjugates 1, 2 and 3 . Yields given are for three steps after HPLC purification.

S. G. Gouin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2684–2688
2687
Centre National de la Recherche Scientifique, the Ministère
Délégué à l’Enseignement Supérieur et à la Recherche.
(a)
(b)
(c)
References and notes
1
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300
400
500
λ (nm)
600
700
800
Figure 3. (a) Excitation (kem = 616 or 545 nm) and emission (kexc = 276 nm) spectra
of 3.Tb (b), 3.Eu (c) in Tris buffer at 298 K.
2
.
Table 1
Absorption maxima wavelengths (kmax in nm) and their corresponding molar
À1
À1
absorption coefficients (
yields ( in %), hydration state (q), brightness (B in M cm ) for the Eu and Tb
glycoconjugates in Tris buffer solution at 298 K (pH 7.4, 50 mM)
e in M cm ), luminescence lifetimes (s in ms), quantum
À1
À1
3+
3+
U
a
a
qb
Bc
Compound
k
max
(
e)
s
H
s
D
U
1
2
3
1
2
3
.Eu
.Eu
.Eu
.Tb
.Tb
.Tb
276 (2420)
276 (2550)
276 (5600)
276 (2355)
276 (2415)
276 (5575)
0.39
0.39
0.39
1.18
1.18
1.18
2.33
2.30
2.35
2.45
2.55
2.46
0.4
0.6
0.6
2.3
2.9
3.0
2.02
2.02
2.03
1.90
1.97
1.90
10
15
34
54
70
3
.
.
Helbich, T. H.; Gossman, A.; Mareski, P. A.; Radüchel, B.; Roberts, T. P. L.;
Shames, D. M.; Mühler, M.; Turetschek, K.; Brasch, R. C. J. Magn. Reson. Imaging
2000, 11, 694.
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Corsi, D. M.; Vander Elst, L.; Muller, R. N.; van Bekkum, H.; Peters, J. A. Chem.
Eur. J. 2001, 7, 64.
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5. (a) Song, Y.; Kohlmeir, E. K.; Meade, T. J. J. Am. Chem. Soc. 2008, 130, 6662; (b)
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P.; Lukeš, I. Chem. Eur. J. 2010, 16, 10094.
a
Data obtained in aerated Tris buffer, H
2
2
O (H) or D O (D) solutions.
b
For europium complexes: q = 1.11(1/
À 1/ À 0.06).
Determined at 276 nm.
H D
s À 1/s À 0.31); for terbium complexes:
6
7
.
.
Tanaka, H.; Ando, Y.; Wada, M.; Takahashi, T. Org. Biomol. Chem. 2005, 3, 3311.
Lemieux, G. A.; Yarema, K. J.; Jacobs, C. L.; Bertozzi, C. R. J. Am. Chem. Soc. 1999,
121, 4278.
q = 5(1/
s
H
s
D
c
8
9
.
.
Bünzli, J.-C. G. Chem. Rev. 2010, 110, 2729.
Bünzli, J.-C. G.; Piguet, C. Chem. Soc. Rev. 2005, 34, 1048.
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Brunet, E.; Juanes, O.; Rodriguez-Ubis, J.-C. Curr. Chem. Biol. 2007, 1, 11.
11. Kropp, J. L.; Windsor, M. W. J. Phys. Chem. 1965, 42, 1599.
2. Laurent, S.; Vander Elst, L.; Wautier, M.; Galaup, C.; Muller, R. N.; Picard, C.
Bioorg. Med. Chem. Lett. 2007, 17, 6230.
3. Pellegatti, L.; Zhang, J.; Drahos, B.; Villette, S.; Suzenet, F.; Guillaumet, G.;
Petoud, S.; Toth, E. Chem. Commun. 2008, 48, 6591.
4. Moats, R. A.; Fraser, S. E.; Meade, T. J. Angew. Chem., Int. Ed. 1997, 36, 726.
5. (a) Mastarone, D. J.; Harrison, V. S. R.; Eckermann, A. L.; Parigi, G.; Luchinat, C.;
Meade, T. J. J. Am. Chem. Soc. 2011, 133, 5329; (b) Floyd, W. C.; Klemm, P. J.;
Smiles, D. E.; Kohlgruber, A. C.; Pierre, V. R. C.; Mynar, J. L.; Freichet, J. M. J.;
Raymond, K. N. J. Am. Chem. Soc. 2011, 133, 2390.
Ln3+ complexation. Meade and co-workers28 previously showed
that such an effect could occur with specific sugar residues and
the Gd ion. They reported that a galactopyranose sugar moiety
tethered to a DO3A-Gd(III) complex could sterically prevent water
access to the Gd ion.
The brightness value, corresponding to the product of the
absorption coefficient at an excitation wavelength and the quan-
tum yield, is indicative of the overall luminescence efficiency of
the complexes. Grafting two pyridine-chelates to a sugar scaffold
enhances the luminescence emission intensity by a factor of 2–3
compared to the corresponding mono-chelate. It seems, therefore,
that the tethered lanthanide complexes do not self-quench in con-
trast to fluorescent organic probes.29 Indeed, two fluorescent
probes in close proximity may self-quench if their absorption
and emission spectra overlap. In this case, the large Stokes shift
of lanthanide complexes overcomes this undesirable self-quench-
ing phenomenon.
1
1
1
1
1
6. Almant, M.; Moreau, V.; Kovensky, J.; Bouckaert, J.; Gouin, S. G. Chem. Eur. J.
2
011, 17, 10029.
17. Maunier, V.; Boullanger, P.; Lafont, D.; Chevalier, Y. Carbohydr. Res. 1997, 299,
9.
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8. (a) Gouin, S. G.; Kovensky, J. Tetrahedron Lett. 2007, 48, 2875; (b) Lyle, F. R. U.S.
Patent 5 973 257, 1985; Chem. Abstr. 1985, 65, 2870.
19. Jiménez-Blanco, J. L.; García Fernández, J. M.; Gadelle, A.; Defaye, J. Carbohydr.
Res. 1997, 303, 367.
2
2
0. Sharbert, B., Eur. Pat. Appl., EP 053313, 1992.
1. Selected spectral data for 11 and 12. Compound 11: NMR 1H (CDCl
3
, 300 MHz)
d (ppm): 1.45 (s, 36H: tBu); 3.53 (s, 8H: CH COO); 4.13 (s, 4H, CH py); 8.11 (s,
2
2
2H, Hpy). NMR 13C (CDCl
, 75 MHz) d (ppm): 28.1 (CH
3
3
2
); 52.5 (NCH COO); 55.8
(
NCH
2
py); 81.4 (Cq tBu); 121.6 (C3,5-py); 142.75 (C4-py); 158.9 (C2,6-py); 167.9
A click methodology was implemented to graft an alkynyl pyr-
idine-chelate to hydrophilic and biocompatible carbohydrate scaf-
folds. The resulting glycoconjugates were shown to retain their
+
+
+
(
1
COOH); 170.5 (COOtBu). MS (ES ) = 660.3 [M+Na ]; 638.1 [M+H ]. Compound
2: NMR 1H (CDCl
, 300 MHz) d (ppm): 1.44 (s, 36H: tBu); 2.03 (t, 1H,
J = 2.4 Hz: HC„); 2.52 (td, 2H, J = 6.6 Hz, J = 2.7 Hz: CH C„); 3.47 (s, 8H:
CH COO); 3.60 (q, 2H, J = 6.6 Hz: CH NH); 4.06 (s, 4H, CH py); 6.94 (m, 1H, NH);
.91 (s, 2H, Hpy). NMR 13C (CDCl
, 75 MHz) d (ppm): 19.3 (CH C„); 28.1 (CH );
8.7(CH NH); 56.0 (NCH COO); 59.8 (NCH py); 70.1 („CH); 81.2 (Cq tBu); 81.4
C„); 118.6 (C3,5-py); 142.7 (C4-py); 159.9 (C2,6-py); 166.2 (CONH); 170.5
3
2
complexing abilities towards Eu3 and Tb with regard to the cor-
+
3+
2
2
2
7
3
3
2
3
responding unfunctionalized chelate. The divalent conjugates 3.Ln
2
2
2
(
Ln = Eu, Tb) were two to three times more fluorescent than their
(CH
COOtBu). MS (ES ) = 711.4 [M+Na ] (100); 689.4 [M+H ] (55) HR-MS (TOF
2
+
+
+
(
monovalent counterparts. Thus, labeling biomolecules with multi-
valent lanthanide probes may offer interesting opportunities to in-
crease the detection threshold of specific targeted biomolecules. In
further investigations, we will explore the relaxivity properties of
the corresponding Gd-conjugates, and apply the synthetic method-
ology developed to the design of carbohydrate lanthanide probes
with higher valency.
+
+
ES ) = calculated for M+H = 689.4126; found = 689.4111.
2. Féau, C.; Klein, E.; Kerth, P.; Lebeau, L. Bioorg. Med. Chem. Lett. 2007, 17, 1499.
2
23. Pérez, D.; Burés, G.; Guitián, E.; Castedo, L. J. Org. Chem. 1996, 61, 1650.
4. (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
2
Ed. 2002, 41, 2596; (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2
002, 67, 3057.
25. Rodios, N. A. J. Heterocycl. Chem. 1984, 21, 1169.
6. Experimental procedure and analytical data for 1. Compound
6.8 mol) and 13 (20.3 mg, 29.4 mol) were dissolved in a dioxane/water
mixture (2/0.5 mL). Copper sulfate (4.2 mg, 26.3 mol) and sodium ascorbate
(10 mg, 54 mol) were added and the mixture was stirred at 70 °C for 45 min
under W irradiation. The mixture was evaporated under reduced pressure,
2
5 (10 mg,
2
l
l
l
Acknowledgments
l
l
dissolved in dichloromethane (10 mL), and the organic layer washed with an
aqueous solution of ethylenediamine tetraacetic acid trisodium salt (500 mg in
This work was carried out with financial support from the
French Agence Nationale de la Recherche (ANR JC07_183019), the
4
10 mL) and water (10 mL). The organic layer was dried over MgSO , filtered

2
688
S. G. Gouin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2684–2688
and evaporated under reduced pressure. The residue was dissolved in a
methanolic solution of ammonia 7 M (5 mL) and the solution was stirred for
h. The solvent was evaporated under reduced pressure and the residue
13C NMR (75 MHz, DMSO): d = 171.4 (CO), 164.5 (NHCO), 156.6 (CPyr), 144.5
(CTrz), 144.0 (CPyr), 121.5 (CHTrz), 120.5 (CPyr), 87.4 (C1), 79.9, 76.9, 72.1, 69.8
3
2 2
(C2,-3,-4,-5), 60.8, 60.2 (C6, CH ), 58.1, 54.4, 25.3 (CH ); HRMS (ES+): found
dissolved in pure TFA (10 mL). The mixture was stirred for 12 h at rt. The
solvent was evaporated and the residue purified by HPLC on a C18 column. The
elution gradient was 100% water—TFA (0.1%) for 10 min then a linear gradient
670.2331 C26 14 requires 670.2320.
36 7
H N O
27. Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle, L.; De
Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin Trans. 2 1999, 493.
28. Urbanczyk-Pearson, L. M.; Femia, F. J.; Smith, J.; Parigi, G.; Duimstra, J. A.;
Eckermann, A. L.; Luchinat, C.; Meade, T. J. Inorg. Chem. 2008, 47, 56.
29. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.;
Molecular Probes, Inc.: Eugene, OR, 1996.
to CH
3%) as a white powder.
= +9 (c = 0.2, H
O); Tr = 18.6 min; 1H NMR (300 MHz, DMSO) d = 9.05 (1H,
br, NH), 8.16 (1H, s, HTrz), 7.90 (2H, s, HPyr), 5.48 (1H, d, J = 9 Hz, H-1), 4.24 (4H,
s, CH ), 2.90 (2H, br, CH NHCO),
N), 3.70–3.20 (18H, m, H-2,-3,-4,-5,-6, 6 Â CH
3
CN 100% for 90 min. The residue was lyophilized to lead to pure 1 (8 mg,
4
[a]
D
2
2
2
2