Chemical synthesis and biological validation of immobilized protein kinase inhibitory Leucettines

Article abstract of DOI:10.1016/j.ejmech.2013.01.035

Leucettines, a family of marine sponge-derived 2-aminoimidazolone alkaloids, are potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). They constitute promising pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. In order to investigate the scope of potential targets of Leucettine L41, a representative member of the chemical class, we designed an affinity chromatography strategy based on agarose-immobilized leucettines. A synthesis protocol for the attachment of a polyethylene (3 or 4 units) linker to L41 was first established. The linker attachment site on L41 was selected on the basis of the co-crystal structure of L41 with several kinases. L41 was then covalently bound to agarose beads through the primary amine located at the end of the linker. Control, kinase inactive Leucettine was also immobilized, as well as free linker devoid of ligand. Extracts of several mouse tissues revealed a complex pattern of interacting proteins, some of which probably resulting from non-specific, hydrophobic binding, while others representing bona fide Leucettine-interacting proteins. DYRK1A and GSK-3 (glycogen synthase kinase-3) were confirmed as interacting targets by Western blotting in various mouse tissues. The Leucettine affinity chromatography resin constitutes a powerful tool to purify and identify the targets of this new promising therapeutic class of molecules.

Full text of DOI:10.1016/j.ejmech.2013.01.035

European Journal of Medicinal Chemistry 62 (2013) 728e737
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chemical synthesis and biological validation of immobilized
protein kinase inhibitory Leucettines
,
Guillaume Burgya b, Tania Tahtouh c, Emilie Durieu c, Béatrice Foll-Josselin c,
,
a,
**
Emmanuelle Limanton a, Laurent Meijer b , François Carreaux
,
*
,
Jean-Pierre Bazureau a
***
a Université de Rennes 1, Institut des Sciences Chimiques de Rennes, ISCR UMR CNRS 6226, Groupe Ingénierie Chimique & Molécules
pour le Vivant (ICMV), Bât. 10A,
Campus de Beaulieu, Avenue du Général Leclerc, CS 74205, 35042 Rennes cedex, Bretagne, France
b ManRos Therapeutics, Perharidy Research Center, Centre de Perharidy, 29680 Roscoff, Bretagne, France
c Station Biologique de Roscoff, CNRS, ‘Protein Phosphorylation and Human Disease’ Group, Place G. Teissier, 29680 Roscoff, Bretagne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Leucettines, a family of marine sponge-derived 2-aminoimidazolone alkaloids, are potent inhibitors of
DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). They
constitute promising pharmacological leads for the treatment of several diseases, including Alzheimer’s
disease and Down syndrome. In order to investigate the scope of potential targets of Leucettine L41,
a representative member of the chemical class, we designed an affinity chromatography strategy based
on agarose-immobilized leucettines. A synthesis protocol for the attachment of a polyethylene (3 or 4
units) linker to L41 was first established. The linker attachment site on L41 was selected on the basis of
the co-crystal structure of L41 with several kinases. L41 was then covalently bound to agarose beads
through the primary amine located at the end of the linker. Control, kinase inactive Leucettine was also
immobilized, as well as free linker devoid of ligand. Extracts of several mouse tissues revealed a complex
pattern of interacting proteins, some of which probably resulting from non-specific, hydrophobic
binding, while others representing bona fide Leucettine-interacting proteins. DYRK1A and GSK-3 (gly-
cogen synthase kinase-3) were confirmed as interacting targets by Western blotting in various mouse
tissues. The Leucettine affinity chromatography resin constitutes a powerful tool to purify and identify
the targets of this new promising therapeutic class of molecules.
Received 8 January 2013
Received in revised form
25 January 2013
Accepted 29 January 2013
Available online 6 February 2013
Keywords:
Leucettamine B
Leucettine
DYRKs
CLKs
GSK-3
Kinase
Kinase inhibitor
Alzheimer’s disease
Pre-mRNA splicing
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
consequently, protein kinases have now become the major class of
screening targets for the pharmaceutical industry. There are cur-
Protein phosphorylation probably constitutes one of the main
intracellular regulation mechanisms. It results from the delicate
balance between the activities of protein kinases and protein
phosphatases. There is constantly growing evidence for links
between abnormal phosphorylation and human disease, and
rently 238 inhibitors of kinases (monoclonal antibodies and low
molecular weight compounds) in clinical trials, mostly targeting
tyrosine kinases and mostly targeting cancer [1].
Among the 518 human kinases, we have focused our efforts on
two classes of serine/threonine kinases, DYRKs (dual-specificity,
tyrosine phosphorylation regulated kinases, 5 members) and
CLKs (cdc2-like kinases, 4 members). These kinases play essential
physiological roles including the regulation of mRNA splicing,
neuronal development, cell death, etc. They appear to be involved
in various diseases including Alzheimer’s disease, Down syndrome
(the DYRK1A gene is located on chromosome 21, and the extra copy
is associated with early signs of Alzheimer’s disease in Down syn-
drome patients), cancer, etc, review in Refs. [2e4]. Only a limited
number of pharmacological inhibitors of DYRKs and CLKs have
been described and characterized [5e11]. Our screening efforts
have led to the identification of a class of inhibitors derived from
Abbreviations: CDKs, cyclin-dependent kinases; CLKs, cdc2-like kinases; DMSO,
dimethylsulfoxide; DTT, dithiothreitol; DYRKs, dual-specificity, tyrosine phosphor-
ylation regulated kinases; EG, ethyleneglycol; GSH, glutathione; GSK-3, glycogen
synthase kinase-3; GST, glutathione-S-transferase; MWI, microwave irradiation;
PBS, phosphate-buffered saline; PEG, polyethylene glycol.
* Corresponding author. Tel.: þ33 (0)2 98 72 94 92.
** Corresponding author. Tel.: þ33 (0)2 23 23 57 34.
*** Corresponding author. Tel.: þ33 (0)2 23 23 66 03.
E-mail addresses: meijer@manros-therapeutics.com(L. Meijer), francois.carreaux@
univ-rennes1.fr (F. Carreaux), jean-pierre.bazureau@univ-rennes1.fr (J.-P. Bazureau).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.035

G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
729
the marine sponge 2-aminoimidazolone alkaloid Leucettamine B
[12, 13]. Starting from this natural product we have synthesized
over 500 derivatives which were collectively named Leucettines.
The first Leucettine derivatives were described by Debdab et al.
[12]. One representative and potent Leucettine, Leucettine L41, was
co-crystallized with CLK3 [12], DYRK1A, DYRK2, CLK1 and PIM1
[13], allowing an excellent understanding of the mode of binding of
these inhibitors to their target kinases.
We have extensivelystudied the selectivityof LeucettineL41 [13].
Results show that Leucettine L41 targets DYRKs and CLKs, but also
interact with a few other kinases such as glycogen synthase kinase-3
(GSK-3). To identify and investigate the full scope of cellular targets
of Leucettine, we have chosen to immobilize Leucettines on agarose
beads and to use this affinity matrix to purifyand identify the targets
of Leucettines from cell lines and animal tissues. This approach
has already been successfully applied to the cyclin-dependent ki-
nase (CDKs) inhibitors purvalanol [14], roscovitine [15] and paul-
lones [16,17]. This article describes the chemical synthesis and
biological validation of immobilized Leucettines and discusses the
further use of this affinity matrix in pharmacological studies.
Our first task was to perform a reliable synthesis of 4-substituted
aniline 9 bearing an azido PEG fragment. Starting from commercial
4-aminophenol 4, a regioselective protection [18] was conducted in
a solution of THF by slow addition of the readily available di-tert-
butyl dicarbonate. After 24 h, thin layer chromatography indicated
consumption of the starting material and a single product 5 was
obtained in quantitative yield (Table 1). Next, the N-t-Boc amino-
phenol 5 was designed to incorporate polyethylene glycol spacer
arms of various lengths, respectively, from 2-[2-(2-chloroethoxy)
ethoxy]ethanol 6a for compound 7a (n ¼ 2) and 2-[2-[2-(2-
hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl-4-
methylbenzenesulfonate 6c for compound 7b (n ¼ 3). Access to
the mono tosylated [19] compound 6c could be easily realized by
reaction of tetraethylene glycol 6b with para-toluene sulfonyl
chloride in dry THF. After reaction (2 h) at 0 ꢀC, the desired com-
pound 6c was obtained in good yield (96%). In the third step, a tosyl
group was introduced on 7(a, b) leading to 8(a, b) in moderate to
good yields (47e75%) according to a modified known procedure
[20]. For the fourth step, our focus was the transformation of the
tosylated compounds 8(a, b) into organic azide by nucleophilic
substitution reaction in the presence of readily available alkali
azides. The choice of this strategy was guided by the interest to use
azides as a potential precursor of amines. Utilization of water as
reaction media [21] in conjunction with microwave dielectric
heating [22] is one of the emerging unconventional methods being
recognized as potential environmentally benign alternatives. The
microwave-assisted reaction of different tosylates with sodium
azide was examined in an aqueous medium [23] without using
a phase-transfer catalyst [24]. Water also offered a safer environ-
mental approach to prevent the potential explosion danger of azi-
dation in a halogenated solvent [25]. The choice of protection/
deprotection strategy is highly important in organic synthesis of
multifunctional molecules and a large variety of protective groups
have been developed along with numerous methods for their
removal. Among them, the tert-butoxycarbonyl (t-Boc) group is
frequently used as a protecting group for amine and many methods
for N-t-Boc deprotection have been reported under acidic condi-
tions [26]. Recently, an elegant catalyst-free water-mediated
deprotection of N-t-Boc aromatic amines has been reported [27].
In this context, our aim for this fourth step was to develop
a convenient and good yielding protocol for introduction of the
azide group on compound 9 associated with N-deprotection using
water under microwave irradiation (MWI). The experiments
allowed defining the optimal reaction conditions for the synthesis
of 9(a, b) by varying reaction temperature, power, ratio of 8 and
sodium azide and composition of the solvent media for N-t-Boc
deprotection under MWI. The optimal reaction conditions were
obtained after 60 min. with a suspension of 8a and two equivalents
of sodium azide in a mixture of EtOH/H2O (1:1) using a closed
reactor to produce 9a (n ¼ 2) in 90% under MWI at 160 ꢀC. In the
same manner, the product 9b (n ¼ 3) was also prepared in good
yield (95%). Next, the second part of the synthesis concerns the
construction of the 2-amino-5-arylidene imidazolin-4-one moiety
bearing the azido polyethylene glycol linker. Access to compound
11 could be accomplished by sulphur/nitrogen displacement of the
SEt group of an imidazolin-4-one 10 with anilines 9(a, b)
substituted in C-4 position by an azido PEG chain. For 10a (R1,
R2 ¼ OCH2O), this compound was directly synthesized according to
our previous method described in literature [12]. A similar strategy
was used for the synthesis of analogue 10b. Starting from com-
mercial thiohydantoine, the (5Z) 5-benzylidene-2-thioxo-imida-
zolin-4-one 13b was easily obtained by Knœvenagel condensation
of benzaldehyde 12b in an AcONa/AcOH media under microwave
conditions of 140 ꢀC for 20 min using a glass tube sealed with
a snap cap (13b: 93% yield). Then 13b underwent S-alkylation with
2. Results
2.1. Chemistry
Leucettine L41 1a and its analogue 1b were synthesized as pre-
viously described [12]. To identify the targets of Leucettine byaffinity
chromatography on agarose beads, we have developed a protocol for
the synthesis of Leucettine L411a and its analogue 1b (Fig.1) bearing
an amino-polyethylene glycol (PEG) linker on the para-position of
the2-phenylaminogroup ofLeucettine. Wereasonedthatadditionof
this linker moiety at this C-4 position might enable us to immobilize
the Leucettine without preventing its kinase inhibitory activity. The
overall synthesis strategy for the target Leucettine derivatives 3(aec)
with an appended PEG linker is outlined in Scheme 1.
Fig. 1. Structures of compounds used. Leucettine L41 (1a), an inactive analogue (1b),
free 3 EG units linker (2), Leucettine L41 with 3 EG units linker (3a), Leucettine L41
with 4 EG units linker (3b), compound 1b with 3 EG units linker (3c).

730
G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
H
H
N
O
N
H2N
O
iv
ii
i
O
O
O
p-MeC6H4SO2Cl
(t-BuO2C)2O
O
OH
OH
OH
O
n
X
OH
6
n
5
4
7a: n = 2
7b: n = 3
6a: n = 2, X = Cl
6b: n = 3, X = OH
6c: n = 3, X = OTs
R1
iii
R2
H
N
H
N
O
H2N
N
v
vi
O
O
O
R1
O
N
O
OTs
O
N3
N3
O
H
n
n
n
O
R2
11a: R1, R2 = OCH2O, n = 2
11b: R1, R2 = OCH2O, n = 3
11c: R1 = R2 = H, n = 2
S
8a: n = 2
8b: n = 3
9a: n = 2
9b: n = 3
N
N
10a: R1, R2 = OCH2O
10b: R1, R2 = H
H
R1
O
R2
H
N
N
N
vii
3a: R1, R2 = OCH2O, n = 2
3b: R1, R2 = OCH2O, n = 3
3c: R1 = R2 = H, n = 2
O
H2N
O
H
PPh3
n
O
R1
R1
R2
R2
H
N
H
R1
R2
S
N
S
S
H
N
viii
ix
O
N
N
H
N
H
O
O
10a: R1, R2 = OCH2O
10b: R1, R2 = H
O
13a: R1, R2 = OCH2O
13b: R1, R2 = H
12a: R1, R2 = OCH2O
12b: R1, R2 = H
thiohydantoin
Scheme 1. Reagents and reaction conditions: (ii) (t-BuO2C)2O, THF, 25 ꢀC, 24 h. (ii) for 7a: 6a, K2CO3 3 equiv., NaI 0.1 eq., MeCN, 80 ꢀC, 48 h; for 7b: 6c, K2CO3 1.5 equiv., DMF, 80 ꢀC,
48 h. (iii) 6b, NaOH aq., p-MeC6H4SO2Cl, THF, 0 ꢀC, 2 h; (iv) 7, p-MeC6H4SO2Cl 2 equiv., Et3N 3 equiv., DMAP 0.2 equiv., CH2Cl2, 25 ꢀC, 24 h; (v) 8, NaN3 2 equiv., EtOHeH2O (1:1),
MWI, 160 ꢀC, 60 min; (vi) 10 1.2 equiv., MWI, 160 ꢀC, 30 min; (vii) Ph3P on polymer, H2O 3 equiv., THF, 70 ꢀC, 48 h; (viii) AcONa 1.1 equiv., AcOH, MWI, 140 ꢀC, 20 min; (ix) EtI
1.1 equiv., K2CO3 0.5 equiv., Me2NCHO, 60 ꢀC, 24 h.
iodoethane (1.1 equiv.) in dry dimethylformamide to give the (5Z)
5-benzylidene-2-ethylsulfanyl-1H-imidazol-4-one 10b in quanti-
tative yield (98%) at 60 ꢀC after a reaction time of 12 h. The struc-
tural identification of this new compound 10b was based on the 1H
and 13C assignments and was performed by extensive 1D and 2D
NMR spectroscopy. 10b was obtained solely as the Z isomer and no
isomerization of the exocyclic double bond was observed during
our synthesis. With the desired 2-ethylsulfanyl-1H-imidazol-4-
ones 10(a,b) in hand, we next examined the sulphur/nitrogen dis-
placement under MWI. Reaction optimization for this reaction
consisted in varying reaction temperature (from 120 to 180 ꢀC),
possible use of a solvent or not, reaction concentration (ratio 9/10
from 1 to 2) and reaction time (from 20 to 40 min). Sulphur/ni-
trogen displacement for the mixture of 9 and 10 (ratio 9a/10a ¼ 1.2)
at 160 ꢀC after 30 min led to the formation of desired product 11a in
the presence of a substantial amount of byproducts probably
resulting from a cleavage of the PEG chain. We observed production
of colored byproducts, possibly caused by difficulties in adequately
controlling the thermal stability of the azido PEG chain on 11a
under solvent-free MWI. Analysis of the byproducts by 1H NMR
suggested cleavage of the PEG chain. Product 11a was purified by
preparative chromatography on silica gel using a step-wise gra-
dient from 100 to 95% of CH2Cl2/MeOH as mobile phase (11a: 16%
yield). In the same manner, compounds 11b and 11c were also
prepared in very low yields (11b, 11c ¼ 6%) with another PEG length
like 12 atom linker with construct 9b or 9 atom linker with con-
struct 9a. For the last step, our focus was the transformation of the
azido PEG imidazol-4-ones 11(aec) into amino imidazol-4-ones
3(aec) with various PEG lengths. The common methods for
reduction of azides are based on hydrides [28] (LiAlH4 in THF),
tin(II) chloride [29] or zinc dust [30] in methanol or palladium-
catalyzed hydrogenolysis [31]. This implies that a specific, tedious
synthetic protocol has to be devised for each new compound. The
so-called Staudinger reaction is a frequently used method [32] for
smooth reduction of azides into amines using triarylphosphanes.
The major drawback of this alternative method is the presence of
free triphenylphosphinoxide that often contaminates the desired
products. Attention was turned towards the choice of an appro-
priate protocol in the preparation of amino partners 3(aec) without
Table 1
Structure and yield of synthesized compounds.
Compound
n
R1, R2
Yielda (%)
5
6c
7a
7b
8a
e
3
2
3
2
3
2
3
e
2
3
2
2
3
2
e
e
98
96
39
68
75
47
90
95
98
18
6
e
e
e
e
8b
9a
e
e
9b
10b
11a
11b
11c
3a
3b
3c
13b
e
H
OCH2O
OCH2O
H
OCH2O
OCH2O
H
6
75
75
76
93
H
a
Isolated yields.

G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
731
byproducts for affinity chromatography. In this context, the use of
polymer-supported triphenylphosphine in the Staudinger reaction
facilitated the final purification, suppressing the formation of free
triphenylphosphinoxide [33]. Finally, treatment of the azido PEG
imidazolin-4-one 11a with 2 equiv. of triphenylphosphine bound to
polymer in THF during 24 h at 81 ꢀC in the presence of water
afforded the amino PEG imidazolin-4-one 3a in 75% yield. In order
to explore the question of generality, the two other amino PEG
imidazolin-4-ones 3(b,c) were synthesized in good yields accord-
ing to the experimental procedure used for 3a; the compounds 3b
and 3c were obtained respectively with an isolated yield of 75 and
76%, respectively (Table 1) and in quantities suitable for affinity
chromatography. The structures of the three (5Z) 2-(4-aminoPEG)
phenylamino-5-arylidene-1H-imidazol-4-ones 3(a-c) were ascer-
tained by high-resolution mass spectrometry, proton and carbon
NMR, confirming that the thermodynamically more stable Z-iso-
mers were obtained.
brains were homogenized and centrifuged and equal quantities of
supernatant (1 mg total protein/10 L beads) were loaded on all
m
four matrices. After 30 min under constant rotation at 4 ꢀC, beads
were carefully washed and bound proteins were analyzed by SDS-
PAGE followed by silver staining and Western blotting using spe-
cific antibodies (Fig. 2). Results show that the absence of any ligand
(with or without linker, Fig. 2, (2) and (-), respectively) provides
excellent control beads which do not bind any protein from mouse
brain. When Leucettines were bound to the matrix, a complex
pattern was observed, whether kinase inactive (3c) or kinase active
(3a, 3b) Leucettines were immobilized (Fig. 2, left panel), sug-
gesting some non-specific, hydrophobic protein binding to the li-
gands. However, when specific Leucettine L41 targets were probed
by Western blotting, DYRK1A and GSK-3 were found to be enriched
on the kinase inhibitor beads, and much less on the control, inactive
Leucettine (Fig. 2, center and right panels). However there was
some modest binding of DYRK1A and GSK-3 to the inactive com-
pound, suggesting either unspecific hydrophobic interactions or
specific, but low affinity, binding, a result that fits with the modest
but detectable effects of inactive Leucettine þ linker on DYRK1A
and GSK-3 (Table 1).
2.2. Biology
Free Leucettine L411a and its analogue 1b, free 3 EG units linker
(2), 3 EG units linker tethered with Leucettine L41 3a or with its
analogue 3c, and 4 EG units linker tethered with Leucettine L41 3b
(Fig. 1A), were first tested at increasing concentrations on a panel of
14 purified protein kinases. IC50 values were determined from the
dose-response curves (Table 2). Results confirm the expected
selectivity of Leucettine L411a towards DYRKs and CLKs, and GSK-3
to a lower extent. Leucettine L41 remained quite a potent inhibitor
even when tethered to a 3 EG 3a or 4 EG units linker 3b (Table 2).
The free 3 EG units linker 2 was completely inactive on the kinases.
Despite the complete lack of activity of the ‘kinase dead’ analogue
1b, the 1b þ linker 3c compound showed very modest but signifi-
cant effects on some kinases, including DYRK1A. These results
confirm the appropriate selection of the linker attachment site on
Leucettine L41 1a, which was based on the co-crystal structure of
Leucettine L41 with various CLKs and DYRKs [12,13]. They also
confirm the key Hydrogen bond between one of the oxygens of the
methylene dioxy and the kinase targets (lack of or very low activity
of 1b without or with the 3 EG units linker, respectively). We thus
have two active ligands (3a, 3b) and two inactive controls 2 and 3c.
All four ligands were immobilized, through the linker primary
We next ran extracts of various mouse tissues on immobilized
Leucettine L41 (3 EG unit linker) (Fig. 3). Silver staining revealed
a different pattern of interacting proteins according to the different
tissues (Fig. 3). Western blot analysis with anti-DYRK1A and anti-
GSK-3 antibodies showed that these kinases are differently
expressed in the different tissues and can be purified and con-
centrated using the Leucettine L41 matrix (Fig. 4).
3. Experimental section
3.1. Chemistry
3.1.1. General
Melting points were determined on a Kofler melting point
apparatus and were uncorrected. 1H NMR spectra were recorded on
BRUKER AC 300 P (300 MHz) spectrometer, 13C NMR spectra on
BRUKER AC 300 P (75 MHz) spectrometer. Chemical shifts are
expressed in parts per million downfield from tetramethylsilane as
an internal standard. Data are given in the following order: d value,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad), number of protons, coupling constants J is given in Hertz.
The mass spectra (HRMS) were taken respectively on a MS/MS
ZABSpec Tof Micromass (EBE TOF geometry) at an ionizing poten-
tial of 8 eV and on a VARIAN MAT 311 at an ionizing potential of
70 eV in the "Centre Régional de Mesures Physiques de l’Ouest"
(CRMPO, Rennes). Reactions under microwave irradiations were
realized in the ExplorerÒ 24 CEM microwave reactor (CEM France)
and also in the Anton Paar Monowave 300Ò microwave reactor
(Anton Paar France) using borosilicate glass vials of 10 or 30 mL
equipped with snap caps (at the end of the irradiation, cooling
reaction was realized by compressed air). The microwave instru-
ment consists of a continuous focused microwave power output
from 0 to 300 W for the ExplorerÒ 24 CEM apparatus and from 0 to
800 W for the Anton Paar Monowave 300Ò apparatus. All the ex-
periments were performed using stirring option. The target tem-
perature was reached with a ramp of 3 min and the chosen
microwave power maintained constant to hold the mixture at this
temperature. The reaction temperature is monitored using cali-
brated infrared sensor and the reaction time included the ramp
period. The microwave irradiation parameters (power and tem-
perature) were monitored by the ChemDriver software package for
the ExplorerÒ 24 CEM apparatus and by the Monowave software
package for the Anton Paar Monowave 300Ò reactor. Preparative
chromatography was realized on a Combi Flash Rf 200 psi (Serlabo
amine, on agarose at a concentration of 12 mmoles/mL of gel as
described in Section 3. We first ran a mouse brain extract on these
affinity matrices and analyzed the bound proteins. Briefly, mouse
Table 2
Protein kinase selectivity of Leucettine L41 (1a), inactive analogue (1b), free linker
(2), Leucettine L41 þ3 EG linker (3a), Leucettine L41 þ4 EG linker (3b), 1b þ 3 EG
linker (3c). Compounds were tested at various concentrations on 14 purified kinases
as described in Section 3. IC50 values, calculated from the doseeresponse curves, are
reported in
inhibitory but IC50 >10
m
M. ꢁ, inactive at the highest concentration tested (10
mM); >10,
m
M.
Kinases
1a
>10
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
1b
2
3a
3b
3c
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
>100
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(100)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
ꢁ(10)
>10
ꢁ(10)
ꢁ(10)
7.4
>10
ꢁ(10)
ꢁ(10)
3.7
ꢁ(10)
ꢁ(10)
ꢁ(10)
>10
CDK9/cyclin T
CK1
d
/ε
>10
>10
ꢁ(10)
CLK1
CLK2
CLK3
CLK4
DYRK1A
DYRK1B
DYRK2
DYRK3
0.035
0.32
3.0
0.031
0.021
0.077
0.22
0.70
0.73
0.075
0.061
0.21
0.55
0.061
0.028
0.072
0.13
0.18
0.20
>10
0.28
0.81
0.081
0.05
0.11
0.15
0.20
0.27
ꢁ(10)
ꢁ(10)
>10
8.5
>10
>10
ꢁ(10)
GSK-3
a
/
b
40

732
G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
Silver Stain
GSK-3
DYRK1A
kDa
191
97
64
51
39
28
19
14
Beads
-
2
3c 3a 3b
-
2
3c 3a 3b
-
2
3c 3a 3b
Fig. 2. Leucettine L41 binding proteins in mouse brain. Extracts, prepared from mouse brain, were loaded on control ethanolamine beads (e), linker 2, inactive analogue 3c beads,
Leucettine L41 3a beads and Leucettine L41 3b beads. After extensive washing, the bound proteins were resolved by SDS-PAGE followed by silver staining (left panel) or Western
blotting with anti-DYRK1A (central panel) and anti-GSK-3 antibodies (right panel).
Technologies France) using pre-packed column of silica gel 60 F 254
Merck equipped with a DAD UV/Vis 200e360 nm detector. Ele-
mental analyses were performed on a Flash Microanalyzer EA1112
CHNS/O Thermo Electron in the "Centre Régional de Mesures
Physiques de l’Ouest" (CRMPO, Rennes). Solvents were evaporated
with a BUCHI rotary evaporator. All usual reagents and solvents
were purchased from Acros, Aldrich Chimie, and Fluka France and
were used without further purification. 2-[2-(2-Aminoethoxy)
ethoxy]ethanol 3 was purchased from Aldrich Chimie France and
compound 10a was prepared according to our previous method
described in literature [12].
CH3), 6.63 (d, 2H, J ¼ 8.5 Hz, H-2), 7.19 (d, 2H, J ¼ 7.9 Hz, H-1), 8.96
(br s, 1H, OH), 9.04 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6)
d
¼ 28.09, 78.49, 114.97, 120.06, 130.88, 152.41, 153.03. HRMS,
m/z ¼ 232.0949 found (calculated for C11H15NO3Na, [M þ Na]þ
requires 232.0949).
3.1.1.2. 2-[2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy]ethyl-4-
methylbenzenesulfonate (6c). In a 100 mL two-necked round-bot-
tomed flask provided with a magnetic stirrer and condenser,
commercial tetraethyleneglycol 6b (22 mL, 0.125 mol, 8 equiv.) was
solubilized in 8 mL of dry THF. Then a solution of sodium hydroxide
(1 g, 25.18 mmol, 1.6 equiv.) in deionized water (6 mL) was added in
the reaction mixture followed by a drop wise addition of a solution
of para-toluene sulfonylchloride (3 g, 15.73 mmol, 1 equiv.) in dry
THF (20 mL). The reaction mixture was vigorously stirred at 0 ꢀC
during 2 h. After addition of cooled water (90 mL), the whole
mixture was transferred into a separating funnel; after separating
and extraction with methylene chloride (3 ꢂ 50 mL), the combined
organic phases were dried over MgSO4, filtered and the solvent of
the filtrate was eliminated in a rotary evaporator under reduced
pressure. The colorless oil was dried under high vacuum (10ꢁ3 Torr)
at 25 ꢀC for 1 h which gave the desired compound 6c in 96% yield
(5.26 g). This product 3c was further used without purification. 1H
3.1.1.1. tert-Butyl N-(4-hydroxyphenyl)carbonate (5). In a 250 mL
two-necked round-bottomed flask provided with a magnetic stirrer
and condenser, commercial 4-aminophenol 4 (4 g, 36.6 mmol) was
solubilized in THF (100 mL) at room temperature. To this mixture,
a solution of commercial di-tert-butyl dicarbonate (8 g, 36.6 mmol)
in THF (50 mL) was added drop wise during 20 min. The reaction
mixture was vigorously stirred at 25 ꢀC during 24 h and was con-
centrated in a rotary evaporator under reduced pressure. The
desired tert-butyl N-(4-hydroxyphenyl)carbamate 5 was obtained
as white needles in quantitative yield (7.58 g) and was further used
without purification. 1H NMR (300 MHz, DMSO-d6)
d
¼ 1.44 (s, 9H,
NMR (300 MHz, CDCl3)
d
¼ 2.45 (s, 3H, CH3), 3.78e3.53 (m, 14H),
Fig. 4. Identification of affinity chromatography purified Leucettine L41 targets from
various mouse tissues. Extracts of mouse tissues (brain, kidney, liver, lung, stomach,
intestine, colon, heart, muscle, spleen, thymus, ovary, testis, skin) were prepared and
loaded on immobilized Leucettine L41 3a (3 EG units linker). Beads were extensively
washed and the bound proteins were resolved by SDS-PAGE followed by Western
blotting using antibodies directed against DYRK1A and GSK-3.
Fig. 3. Affinity chromatography purification of Leucettine L41 targets from various
mouse tissues. Extracts of mouse tissues (brain, kidney, liver, lung, stomach, intestine,
colon, heart, muscle, spleen, thymus, ovary, testis, skin) were prepared and loaded on
immobilized Leucettine L41 3a (3 EG units linker). Beads were extensively washed and
the bound proteins were resolved by SDS-PAGE followed by silver staining.

G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
733
4.16 (t, 2H, J ¼ 5.4 Hz, H-10), 7.34 (d, 2H, J ¼ 8.6, 0.6 Hz, H-1), 7.80 (d,
and condenser, a mixture of carbamate 7a (2.3 g, 6.72 mmol)
with 4-dimethylaminopyridine (136 mg, 1.34 mmol, 0.2 equiv.)
and triethylamine (2.32 mL, 20.15 mmol, 3 equiv.) in methylene
chloride (30 mL) was stirred at 0 ꢀC during 30 min. To this cooled
reaction mixture was added dropwise a solution of p-tolulene-
sulfonylchloride (2.65 g, 16.79 mmol, 2.5 equiv.) during 20 min. The
reaction mixture was stirred vigorously at room temperature dur-
ing 24 h. After elimination of the solvent in a rotary evaporator
under reduced pressure, the crude residue was submitted to puri-
fication by preparative chromatography (Combi Flash Rf 200 psi
apparatus) on pre-packed column of silica gel 60F 254 Merck using
a step-wise gradient of cyclohexane/AcOEt (100e50%). Pooling and
evaporation of the eluent in vacuum gave the expected compound
8a in 75% yield (2.49 g) as mobile colorless oil. 1H NMR (300 MHz,
2H, J ¼ 8.3 Hz, H-2). 13C NMR (75 MHz, CDCl3)
d
¼ 21.59, 61.60,
68.61, 69.27, 70.23, 70.37, 70.57, 70.64, 72.47, 127.91, 129.82, 132.88,
144.84. HRMS, m/z ¼ 371.1144 found (calculated for C15H24O7SNa,
[M þ Na]þ requires 371.1140).
3.1.1.3. tert-Butyl N-[4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]phe-
nyl]carbamate (7a). In a 250 mL two-necked round-bottomed flask
provided with a magnetic stirrer and condenser, tert-butyl N-(4-
hydroxyphenyl)carbamate 5 (1 g, 4.78 mmol) was dissolved in dry
acetonitrile (10 mL). To this solution was added successively at
room temperature 2-[2-(2-chloroethoxy)ethoxy]ethanol 6a
(1.4 mL, 9.56 mmol, 2 equiv.) and potassium carbonate (1.98 g,
14.34 mmol, 3 equiv.). The reaction mixture was vigorously stirred
at 81 ꢀC during 48 h. After cooling down to room temperature, the
solvent was eliminated in a rotary evaporator under reduced
pressure and to the crude residue was added 50 mL of water. The
whole mixture was transferred to a separating funnel; after sepa-
rating and extraction with AcOEt (3 ꢂ 50 mL), the combined
organic phases were dried over MgSO4, filtered and the solvent of
the filtrate was eliminated in a rotary evaporator under reduced
pressure. The crude residue was submitted to purification by pre-
parative chromatography (Combi Flash Rf 200 psi apparatus) on
pre-packed column of silica gel 60F 254 Merck using a step-wise
gradient of cyclohexane/AcOEt (100e50%). Pooling and evapo-
ration of the eluent in vacuum gave the expected compound 7a in
39% yield (636 mg) as mobile colorless oil. 1H NMR (300 MHz,
DMSO-d6)
d
¼ 1.43 (s, 9H, 3 ꢂ CH3), 2.35 (s, 3H, CH3), 3.52e3.63 (m,
6H, H-4, H-5, H-6), 3.72 (t, 2H, J ¼ 5.5 Hz, H-7), 3.99 (t, 2H,
J ¼ 5.5 Hz, H-3), 4.07 (t, 2H, J ¼ 7.5 Hz, H-8), 6.35 (br s, 1H, NH), 6.76
(d, 2H, J ¼ 11 Hz, H-2), 7.18 (d, 1H, J ¼ 10 Hz, 1H, H-10), 7.26 (d, 2H,
J ¼ 11 Hz, H-1), 7.72 (d, 2H, J ¼ 11.0 Hz, H-9). 13C NMR (75 MHz,
DMSO-d6)
d
¼ 21.62 (CH3), 28.35 (3 ꢂ CH3), 67.72 (CH2), 68.70 (CH2),
69.24 (CH2), 69.82 (CH2), 70.72 (CH2), 70.77 (CH2), 115.01 (CH, Ar),
120.43 (CH, Ar), 127.96 (CH, Ar), 129.82 (CH, Ar), 131.69, 132.96,
144.81 (CeS), 153.13 (CeO), 154.76 (CeN), 171.25 (C]O). HRMS,
m/z ¼ 518.1826 found (calculated for C24H33NO8Na, [M þ Na]þ
requires 518.1824).
3.1.1.6. 2-[2-[2-[2-[4-(tert-Butoxycarbonylamino)phenoxy]ethoxy]
DMSO-d6)
d
¼ 1.48 (s, 9H, CH3), 3.77e3.53 (m, 10H, H-3, H-4, H-5,
ethoxy]ethoxy]ethyl-4-methylbenzenesulfonate (8b). In a 50 mL
H-6, H-7), 3.81 (t, 2H, H-8), 6.34 (br s, 1H, NH), 6.82 (d, 2H,
two-necked round-bottomed flask provided with a magnetic stirrer
and condenser, a solution of p-toluenesulfonylchloride (9.26 g,
48.61 mmol, 1.5 equiv.) in methylene chloride (10 mL) was added
dropwise during 20 min. in a mixture at 0 ꢀC of tert-butyl N-[4-[2-
[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]phenyl]carbamate
7b (12.52 g, 32.5 mmol, 1 equiv.) and triethylamine (9 mL,
97.52 mmol, 3 equiv.) in 30 mL of methylene chloride. The reaction
mixture was stirred vigorously at room temperature during 24 h.
After elimination of the solvent in a rotary evaporator under
reduced pressure, the crude residue was submitted to purification
by preparative chromatography (Combi Flash Rf 200 psi apparatus)
on pre-packed column of silica gel 60F 254 Merck using a step-wise
gradient of cyclohexane/AcOEt (100e50%). Pooling and evaporation
of the eluent in vacuum gave the expected compound 8b in 47%
yield (8.24 g) as mobile colorless oil. 1H NMR (300 MHz, DMSO-d6)
J ¼ 8.9 Hz, H-2), 7.23 (d, 2H, J ¼ 8.5 Hz, H-1). 13C NMR (75 MHz,
DMSO-d6)
d
¼ 28.35, 61.66, 67.65, 69.73, 70.32, 70.75, 72.49
(3 ꢂ CH3), 114.98 (C-2), 120.44 (C-1), 131.78 (CMe3), 153.20 (CeO),
154.65 (CeN), 171.20 (C]O). HRMS, m/z ¼ 364.1736 found (calcu-
lated for C17H27NO6Na, [M þ Na]þ requires 364.1736).
3.1.1.4. tert-Butyl
N-[4-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]
ethoxy]phenyl]carbamate (7b). In a 50 mL two-necked round-bot-
tomed flask provided with a magnetic stirrer and condenser was
added successively 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]
ethoxy]ethyl-4-methylbenzenesulfonate 6c (3.03 g, 8.65 mmol,
1 equiv.), potassium carbonate (1.98 g, 12.97 mmol, 1.5 equiv.) and
tert-butyl N-(4-hydroxyphenyl)carbamate 5 (1.99 g, 9.51 mmol,
1.1 equiv.) in 15 mL of dry THF. The reaction mixture was vigorously
stirred at 80 ꢀC during 48 h. After cooling down to room tempera-
ture, the solvent was eliminated in a rotary evaporator under
reduced pressure and to the crude residue was added 50 mL of
water. The whole mixture was transferred to a separating funnel;
after separating and extraction with methylene chloride
(2 ꢂ 100 mL), the combined organic phases were dried over MgSO4,
filtered and the solvent of the filtrate was eliminated in a rotary
evaporator under reduced pressure. The crude residue was sub-
mitted to purification by preparative chromatography (Combi Flash
Rf 200 psi apparatus) on pre-packed column of silica gel 60F 254
Merck using a step-wise gradient of cyclohexane/AcOEt (100e50%).
Pooling and evaporation of the eluent in vacuum gave the expected
compound 7b in 68% yield (2.27 g) as mobile colorless oil. 1H NMR
d
¼ 1.44 (s, 9H), 2.38 (s, 3H, CH3), 3.62e3.46 (m, 8H), 3.76e3.62 (m,
2H), 4.03e3.93 (m, 2H), 4.15e4.03 (m, 4H), 6.80 (d, 2H, J ¼ 8.5 Hz,
Ar), 7.33 (d, 2H, J ¼ 8.6 Hz, Ar), 7.44 (d, 2H, J ¼ 8.4 Hz, Ar), 7.75 (d,
2H, J ¼ 8.4 Hz, Ar), 9.12 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6)
d
¼ 153.57, 152.87, 144.82, 132.62, 132.31, 130.05, 127.55, 119.54,
114.36, 69.90, 69.81, 69.73, 69.63, 69.59, 68.94, 67.80, 67.15, 28.08,
26.27, 20.99 HRMS, m/z ¼ 562.2086 found (calculated for
C26H37NO9SNa, [M þ Na]þ requires 562.2086).
3.1.1.7. 4-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]aniline
(9a). In
a 30 mL glass tube were placed successively the compound 2-[2-[2-
[4-(tert-butoxycarbonylamino)phenoxy]ethoxy]ethoxy]ethyl-
methylbenzenesulfonate 8a (2.5 g, 5.04 mmol) and sodium azide
(655 mg, 10.08 mmol, 2 equiv.) in 20 mL of a solution EtOH/H2O
(1:1). The glass tube was sealed with a snap cap and placed in the
Anton Paar MonowaveÒ 300 microwave cavity (P ¼ 800 W). The
stirred mixture was irradiated at 160 ꢀC for 60 min. After micro-
wave dielectric heating, the crude reaction mixture was allowed to
cool down at room temperature and then was concentrated in
a rotary evaporator under reduced pressure. After extraction with
AcOEt (3 ꢂ 50 mL) and pooling, the combined phases were con-
centrated in vacuum and the resulting crude residue was dried
(300 MHz, CDCl3)
d
¼ 1.51 (s, 9H, 3 ꢂ CH3), 3.62 (t, 2H, J ¼ 5.0 Hz),
3.78e3.66 (m, 12H), 3.84 (t, 2H), 4.11 (t, 2H, J ¼ 8.6, 6.0 Hz, H-10),
6.35 (br s, 1H, NH), 6.86 (d, 2H, J ¼ 9.0 Hz, H-2), 7.25 (d, 2H,
J ¼ 9.5 Hz, H-1). HRMS, m/z ¼ 408.2000 found (calculated for
C19H31NO7Na, [M þ Na]þ requires 408.1999).
3.1.1.5. 2-[2-[2-[4-(tert-Butoxycarbonylamino)phenoxy]ethoxy]
ethoxy]ethyl-4-methylbenzenesulfonate (8a). In
necked round-bottomed flask provided with a magnetic stirrer
a 250 mL two-

734
G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
under high vacuum (10ꢁ3 Torr) at 25 ꢀC for 1 h which gave
the desired 4-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]aniline 9a as
a brown viscous oil in 90% yield (1.21 g). This product was further
was heated at 60 ꢀC under vigorous stirring during 12 h. After cool-
ing down to room temperature, 30 mL of deionized water were
added in one portion at 0 ꢀC in the reaction mixture. The resulting
suspension was stirred slowly at 0 ꢀC over a period of 2 h and the
desired insoluble (5Z) 5-benzylidene-2-ethylsulfanyl-1H-imidazol-
4-one 10b was collected by filtration and washed twice with
deionized water (10 mL). Then, the compound 10b was dried under
high vacuum (10ꢁ2 Torr) at 25 ꢀC for 1 h and was further used
without purification. The product 10b was obtained as yellow nee-
dles in 98% yield (1.81 g). Mp >250 ꢀC. 1H NMR (300 MHz, DMSO-d6)
used without purification. 1H NMR (300 MHz, DMSO-d6)
d
¼ 3.78e
3.63 (m, 6H, H-4, H-5, H-6), 3.39 (t, 2H, J ¼ 5.6 Hz, H-7), 3.83 (t, 2H,
J ¼ 5.6 Hz, H-8), 4.06 (t, 2H, J ¼ 5.6 Hz, H-3), 6.63 (d, 2H, J ¼ 8.9 Hz,
H-1), 6,77 (d, 2H, J ¼ 8.9 Hz, H-2). 13C NMR (75 MHz, DMSO-d6)
d
¼ 68.13 (CH2), 70.01 (CH2), 70.06 (CH2), 70.73 (CH2), 70.72 (CH2),
70.81 (CH2), 115.87 (CH], Ar), 116.33 (CH], Ar), 140.19 (CeO),
151.91 (CeN). HRMS, m/z ¼ 289.1274 found (calculated for
C12H18N4O3Na, [M þ Na]þ requires 289.1276).
d
¼ 1.39 (t, 3H, CH3), 3.28 (q, 2H, CH2), 6.73 (s, 1H, CH]), 7.30e7.52
(m, 3H, H-3, H-4, H-5, Ar), 8.17 (d, 2H, H-2, H6, Ar), 11.78 (br s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6)
3.1.1.8. 4-[2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethoxy]aniline
(9b). In a 30 mL glass tube were placed successively the compound
2-[2-[2-[2-[4-(tert-butoxycarbonylamino)phenoxy]ethoxy]ethoxy]
ethoxy]ethyl-4-methylbenzenesulfonate 8b (2.5 g, 4.63 mmol,
1 equiv.) and and sodium azide (655 mg, 10.08 mmol, 2 equiv.) in
20 mL of a solution EtOH/H2O (1:1). The glass tube was sealed with
a snap cap and placed in the Anton Paar MonowaveÒ 300 micro-
wave cavity (P ¼ 800 W). The stirred mixture was irradiated at
160 ꢀC for 60 min. After microwave dielectric heating, the crude
reaction mixture was allowed to cool down at room temperature
and then, was concentrated in a rotary evaporator under reduced
pressure. After extraction with AcOEt (3 ꢂ 50 mL) and pooling, the
combined phases were concentrated in vacuum and the resulting
crude residue was dried under high vacuum (10ꢁ3 Torr) at 25 ꢀC for
1 h which gave the desired 4-[2-[2-[2-(2-azidoethoxy)ethoxy]
ethoxy]ethoxy]aniline 9b as a brown viscous oil in 95% yield
(1.36 g). This product was further used without purification. 1H
d
¼ 14.51 (CH2), 24.20 (CH3),
120.60 (CH]), 128.60 (C-3, C-5), 129.43 (C-4), 131.32 (C-2, C-6),
134.31 (C-1), 139.24 (CeN), 164.44 (C]O), 170.52 (CeS). HRMS,
m/z ¼ 255.0569 found (calculated for C12H12N2OSNa, [M þ Na]þ
requires 255.0568).
3.1.1.10. (5Z)
2-[4-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]phenyl-
amino]-5-(benzo[1,3]dioxol-5-ylmethylene)-3,5-dihydro-imidazol-4-
one (11a). In a 10 mL glass tube were placed successively the
compound (5Z) 5-benzo[1,3]dioxol-5-ylmethylene-2-ethylsulfanyl-
3,5-dihydro-imidazol-4-one 10a (100 mg, 0.36 mmol, 1 equiv.)
and 4-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]aniline 9a (140 mg,
0.72 mmol, 1.4 equiv.). The glass tube was sealed with a snap cap
and placed in the ExplorerÒ 24 CEM microwave cavity (P ¼ 30 W).
The stirred mixture was irradiated at 160 ꢀC for 30 min. After mi-
crowave dielectric heating, the crude reaction mixture was allowed
to cool down at room temperature. The residue was dissolved in
methylene chloride (5 mL) and was submitted to purification by
preparative chromatography (Combi Flash Rf 200 psi apparatus) on
pre-packed column of silica gel 60F 254 Merck using a step-wise
gradient of CH2Cl2/MeOH (100e95%). Pooling and evaporation of
the eluent in vacuum gave the desired azido compound 11a in 18%
yield (25.6 mg) as a yellowish powder. Mp >260 ꢀC (decom-
NMR (300 MHz, DMSO-d6)
d
¼ 3.66e3.29 (m, 12H), 3.71e3.67 (m,
2H), 3.95e3.84 (m, 2H), 6.49 (d, 2H, J ¼ 8.6 Hz, Ar), 6.63 (d, 2H,
J ¼ 8.6 Hz, Ar). 13C NMR (75 MHz, DMSO-d6v
)
d
¼ 149.70, 142.37,
115.25, 114.89, 69.82, 69.78, 69.75, 69.63, 69.19, 69.11, 67.49, 49.92.
HRMS, m/z ¼ 333.1537 found (calculated for C14H22N4O4Na,
[M þ Na]þ requires 333.1538).
position). 1H NMR (300 MHz, DMSO-d6)
d
¼ 3.39 (t, 2H, J ¼ 5.4 Hz),
3.1.1.9. (5Z) 5-Benzylidene-2-ethylsulfanyl-3,5-dihydro-imidazol-4-
one (10b). In a 30 mL glass tube were placed successively com-
mercial thiohydantoin (1 g, 8.61 mmol), 0.96 mL of benzaldehyde
12b (9.47 mmol, 1.1 equiv.), sodium acetate (777 mg, 9.47 mmol,
1.1 equiv.) and 4 mL of glacial acetic acid. The glass tube was sealed
with a snap cap and placed in the ExplorerÒ 24 CEM microwave
cavity (P ¼ 300 W). The stirred mixture was irradiated at 140 ꢀC for
20 min under vigorous stirring. After microwave dielectric heating,
the crude reaction mixture was allowed to cool down at room
temperature after which 10 mL of deionized water were added in
one portion. Then, the resulting mixture was triturated during
20 min. with slow stirring and yellow needles appeared in the
suspension. The desired insoluble (5Z) 5-benzylidene-2-thioxo-
imidazolidin-4-one 13b was collected by filtration then washed
with deionized water (2 mL) and dried under high vacuum
(10ꢁ2 Torr) at 25 ꢀC for 1 h. The product 13b was obtained as yellow
needles in 93% yield (1.63 g). Mp >250 ꢀC. 1H NMR (300 MHz,
3.68e3.54 (m, 6H), 3.74 (t, 2H, J ¼ 5.6 Hz, H-13), 4.08 (t, 2H,
J ¼ 4.8 Hz, H-14), 6.05 (s, 2H, OCH2O), 6.41 (s, 1H, CH]), 6.94 (dd,
3H, J ¼ 8.3 Hz, H-7, H-6), 7.43 (d, 1H, J ¼ 8.2 Hz, H-5), 7.64 (d, 2H,
J ¼ 8.9 Hz, H-8), 7.90 (s, 1H, H-2), 9.60 (br s, 1H, NH), 10.62 (br s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6)
d
¼ 49.93 (OCH2O), 67.30 (CH2),
68.98 (CH2), 69.23 (CH2), 69.65 (CH2), 69.89 (CH2), 108.37 (CH]),
109.17 (CH]), 113.96 (CH]), 114.64 (CH]), 120.98 (CH]C), 125.32
(CH]C), 129.91, 139.21, 146.99, 147.29, 154.29, 154.53, 170.34.
HRMS, m/z ¼ 481.1837 found (calculated for C23H24N6O6Na,
[M þ Na]þ requires 481.1835).
3.1.1.11. (5Z)
2-[4-[2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethoxy]
phenylamino]-5-(benzo[1,3]dioxol-5-ylmethylene)-3,5-dihydro-imi-
dazol-5-one (11b). In a 10 mL glass tube were placed successively
the
compound
(5Z)
5-benzo[1,3]dioxol-5-ylmethylene-2-
ethylsulfanyl-3,5-dihydro-imidazol-4-one 10a (81 mg, 0.293 mmol,
1 equiv.) then 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]an-
iline 9b (100 mg, 0.351 mmol, 1.2 equiv.) and 10 mL of hexane. The
glass tube was sealed with a snap cap and placed in the ExplorerÒ 24
CEM microwave cavity (P ¼ 300 W). The stirred mixture was irradi-
ated at 160 ꢀC for 30 min. After microwave dielectric heating, the
crude reaction mixture was allowed to cool down at room temper-
ature. The residue was dissolved in methylene chloride (5 mL)
and was submitted to purification by preparative chromatography
(Combi Flash Rf 200 psi apparatus) on pre-packed column of silica
gel 60F 254 Merck using a step-wise gradient of CH2Cl2/MeOH (100e
95%). Pooling and evaporation of the eluent in vacuum gave the
desired azido compound 11b in 6% yield (8.8 mg) as a yellowish
powder. Mp >260 ꢀC (decomposition). 1H NMR (300 MHz, DMSO-d6)
DMSO-d6)
7.73 (d, 2H, J ¼ 4.1 Hz, H-2, H-6), 12.15 (br s, 1H, NH), 12.38 (br s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6)
d
¼ 6.48 (s, 1H, CH]), 7.51e7.30 (m, 3H, H-3, H-4, H-5),
d
¼ 111.50 (CH]), 127.68 (C-1),
128.74 (C-3, C-5), 129.19 (C-4), 130.13 (C-2, C-6), 132.27 (CeN),
165.74 (C]O), 179.18 (C]S). HRMS, m/z ¼ 227.0251 found (calcu-
lated for C10H8N2OSNa, [M þ Na]þ requires 227.0250).
In a 50 mL two-necked round-bottomed flask provided with
a magnetic stirrer and condenser were placed 0.70 mL of ethyl iodide
(8.78 mmol, 1.1 equiv.) and potassium carbonate (551 mg,
3.99 mmol, 0.5 equiv.). To this mixture was added a solution of (5Z)
5-benzylidene-2-thioxo-imidazolidin-4-one 13b (1.63 g, 7.98 mmol,
1 equiv.) in dry dimethylformamide (10 mL). The reaction mixture

G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
735
d
¼ 3.63e3.39 (m, 12H), 3.81e3.67 (m, 3H), 4.15e3.98 (m, 2H), 6.04
400 Mesh, Sigma) and (5Z) 2-[4-[2-[2-[2-(2-azidoethoxy)ethoxy]
ethoxy]ethoxy]phenylamino]-5-benzo[1,3]dioxol-5-ylmethylene-
3,5-dihydro-imidazol-5-one 11b (98 mg, 0.187 mmol) in 5 mL of
THF was heated under reflux during 24 h in a thermostated oil bath.
To this mixture was added deionized water (0.5 mL) and heating
was pursued at 81 ꢀC for 24 h under magnetic stirring. After cooling
down to room temperature, insoluble triphenylphosphine oxide
bound on polymer was collected by filtration then washed with
ethanol (2 ꢂ 10 mL). The resulting filtrate was concentrated in
a rotary evaporator under reduced pressure and the crude residue
was dried under high vacuum (10ꢁ3 Torr) at 25 ꢀC for 1 h which
gave a yellowish solid compound 3b in 75% yield (73 mg). This
product 3b was further used without purification for immobiliza-
tion on Sepharose beads. Mp >260 ꢀC (decomposition). 1H NMR
(s, 2H), 6.41 (s, 1H, CH]), 7.06e6.86 (m, 3H), 7.43 (d, 1H, J ¼ 8.2 Hz,
Ar), 7.64 (d, 2H, J ¼ 8.9 Hz, Ar), 7.90 (s, 1H, H-2), 9.60 (br s, 1H, NH),
10.62 (br s, 1H, NH). HRMS, m/z ¼ 521.2025 found (calculated for
C25H30N4O7Na, [M þ Na]þ requires 521.2012).
3.1.1.12. (5Z) 2-[4-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]anilino]-4-
benzylidene-3,5-dihydro-imidazol-5-one (11c). In
a 10 mL glass
tube were placed successively the compound (5Z) 5-benzylidene-2-
ethylsulfanyl-3,5-dihydro-imidazol-4-one 10b (100 mg, 0.43 mmol,
1 equiv.) and 4-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]aniline 9a
(137 mg, 0.52 mmol, 1.2 equiv.). The glass tube was sealed with
a snap cap and placed in the ExplorerÒ 24 CEM microwave cavity
(P ¼ 300 W). The stirred mixture was irradiated at 160 ꢀC for 30 min.
After microwave dielectric heating, the crude reaction mixture was
allowed to cool down at room temperature. The residue was dis-
solved in methylene chloride (5 mL) and was submitted to purifi-
cation by preparative chromatography (Combi Flash Rf 200 psi
apparatus) on pre-packed column of silica gel 60F 254 Merck using
a step-wise gradient of CH2Cl2/MeOH (100e95%). Pooling and
evaporation of the eluent in vacuum gave the desired azido com-
pound 11c in 6% yield (11.3 mg) as a yellowish powder. Mp >260 ꢀC
(300 MHz, DMSO-d6)
d
¼ 4.00e3.05 (m, 12H), 4.26e4.03 (m, 4H),
6.11 (s, 2H, OCH2O), 6.46 (s, 1H, CH]), 6.82e7.08 (m, 3H, Ar), 7.47
(d,1H, J ¼ 8.3 Hz, Ar), 7.72 (d, 2H, J ¼ 8.4 Hz, Ar), 7.94 (s, 1H), 9.62 (br
s, 1H, NH), 9.74 (br s, 2H, NH2), 10.66 (br s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6)
d
¼ 154.16, 147.30, 146.87, 132.12, 129.94,
125.18, 120.85, 114.62, 109.06, 108.38, 101.06, 69.85, 69.74, 69.68,
69.53, 68.94, 67.28. HRMS, m/z ¼ 521.2025 found (calculated for
C25H30N4O7Na, [M þ H]þ requires 521.2012).
(decomposition). 1H NMR (300 MHz, DMSO-d6)
d
¼ 3.38 (t, 2H,
3.1.1.15. (5Z) 2-[4-[2-[2-(2-Aminoethoxy)ethoxy]ethoxy]anilino]-4-
benzylidene-3,5-dihydro-imidazol-5-one (3c). In a 10 mL round-
bottomed flask provided with a magnetic stirrer and condenser,
a mixture of 100 mg (2 equiv.) of triphenylphosphine (1.5 mmol/g)
bounded on polymer (200e400 Mesh, Sigma) and (5Z) 2-[4-[2-[2-
(2-azidoethoxy)ethoxy]ethoxy]anilino]-4-benzylidene-3,5-dihydro-
imidazol-5-one 11c (42 mg, 0.096 mmol) in 5 mL of THF was heated
under reflux during 24 h in a thermostated oil bath. To this mixture
was added deionized water (0.5 mL) and heating was pursued at
81 ꢀC during 24 h under magnetic stirring. After cooling down to
room temperature, insoluble triphenylphosphine oxide bound on
polymer was collected by filtration then washed with ethanol
(2 ꢂ 10 mL). The resulting filtrate was concentrated in a rotary
evaporator under reduced pressure and the crude residue was dried
under high vacuum (10ꢁ3 Torr) at 25 ꢀC for 1 h which gave a yel-
lowish solid compound 3c in 76% yield (32 mg). This product 3c was
further used without purification for immobilization on Sepharose
beads. Mp >260 ꢀC (decomposition). 1H NMR (300 MHz, DMSO-d6)
J ¼ 4.8 Hz), 3.70e3.52 (m, 6H), 3.76 (t, 2H, J ¼ 4.3 Hz), 4.09 (t, 2H,
J ¼ 4.8 Hz), 6.44 (s,1H, CH]), 6.98 (d, 2H, J ¼ 8.8 Hz, H-8), 7.28 (d,1H,
J ¼ 7.2 Hz, 1H), 7.39 (dd, 2H, J ¼ 7.4 Hz, H-3, H-5), 7.69 (d, 2H,
J ¼ 8.3 Hz, H-7), 8.07 (d, 2H, J ¼ 7.5 Hz, H-6, H-2), 9.69 (br s, 1H, NH),
10.67 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6)
d
¼ 49.93, 67.27,
68.97, 69.23, 69.66, 69.89, 113.49, 114.70, 121.03, 127.62, 128.38,
130.08, 131.84, 135.54, 140.81, 154.33, 155.06, 170.44. HRMS,
m/z ¼ 459.1759 found (calculated for C22H24N6O4Na, [M þ Na]þ
requires 459.1759).
3.1.1.13. (5Z)
2-[4-[2-[2-(2-Aminoethoxy)ethoxy]ethoxy]phenyl-
amino]-5-(benzo[1,3]dioxol-5-ylmethylene)-3,5-dihydro-imidazol-4-
one (3a). In a 10 mL round-bottomed flask provided with a magnetic
stirrer and condenser, a mixture of 175 mg (2 equiv.) of triphenyl-
phosphine (1.5 mmol/g) bounded on polymer (200e400 Mesh,
Sigma) and (5Z) 2-[4-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]phenyl-
amino]-5-benzo[1,3]dioxol-5-ylmethylene-3,5-dihydro-imidazol-4-
one 11a (125 mg, 0.26 mmol) in 5 mL of THF was heated under reflux
during 24 h in a thermostated oil bath. To this mixture was added
deionized water (0.5 mL) and heating was pursued at 81 ꢀC for 24 h
under magnetic stirring. After cooling down to room temperature,
insoluble triphenylphosphine oxide bound on polymer was collected
by filtration then washed with ethanol (2 ꢂ10 mL). The resulting
filtrate was concentrated in a rotary evaporator under reduced
pressure and the crude residue was dried under high vacuum
(10ꢁ3 Torr) at 25 ꢀC for 1 h which gave a yellowish solid compound
3a in 75% yield (88.6 mg). This product 3a was further used without
purification for immobilization on Sepharose beads. Mp >260 ꢀC
d
¼ 3.75e3.56 (m, 8H), 3.81 (t, 2H, J ¼ 4.6 Hz), 4.16 (t, 2H, J ¼ 5.0 Hz),
6.48 (s, 1H, CH]), 7.04 (d, 2H, J ¼ 9.1 Hz, H-8), 7.33 (d, 1H, J ¼ 7.3 Hz,
H-4), 7.47 (dd, 2H, J ¼ 7.6 Hz, H-3, H-5), 7.78 (d, 2H, J ¼ 8.9 Hz, H-7),
8.11 (d, 2H, J ¼ 7.6 Hz Hz, H-2, H-6), 9.60 (br s,1H, NH), 9.71 (br s, 2H,
NH2), 10.51 (br s, 1H, NH). HRMS, m/z ¼ 433.1850 found (calculated
for C22H26N4O4Na, [M þ H]þ requires 433.1860).
3.2. Biology
3.2.1. Buffers
(decomposition). 1H NMR (300 MHz, DMSO-d6)
d
¼ 3.40 (t, 2H,
Bead buffer: 50 mM Tris (pH 7.4), 5 mM NaF, 250 mM NaCl,
5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40 and protease inhibitor
cocktail.
J ¼ 5.1 Hz), 3.67e3.54 (m, 6H), 3.74 (t, 2H, J ¼ 4.6 Hz, H-13), 4.07 (t,
2H, J ¼ 4.6 Hz, H-14), 6.05 (s, 2H, OCH2O), 6.40 (s, 1H, CH]), 6.94 (dd,
3H, J ¼ 8.5 Hz, H-6, H-8), 7.44 (d, 1H, J ¼ 7.9 Hz, H-5), 7.63 (d, 2H,
J ¼ 9 Hz, H-7), 7.87 (s, 1H, H-2), 9.61 (br s, 1H, NH), 9.72 (br s, 2H,
NH2), 10.62 (br s, 1H, NH). HRMS, m/z ¼ 455.1931 found (calculated
for C23H27N4O6, [M þ H]þ requires 455.1930).
Blocking buffer: 1 M ethanolamine in coupling buffer, pH 8.0.
Coupling buffer: 0.1 M NaHCO3 and 0.2 M NaCl, pH 8.3.
Homogenization buffer: 60 mM
b-glycerophosphate, 15 mM
p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 15 mM EGTA,
15 mM MgCl2, 2 mM dithiothreitol, 1 mM sodium orthovanadate,
1 mM sodium fluoride, 1 mM phenylphosphate disodium and
protease inhibitor cocktail.
Washing buffer: 0.1 M CH3COONa, pH 4.
All chemicals were purchased from Sigma, unless otherwise
stated and the protease inhibitor cocktail was from Roche.
3.1.1.14. (5Z)-2-[4-[2-[2-[2-(2-Aminoethoxy)ethoxy]ethoxy]ethoxy]
phenylamino]-5-(benzo[1,3]dioxol-5-ylmethylene)-3,5-dihydro-imi-
dazol-4-one (3b). In a 10 mL round-bottomed flask provided with
a magnetic stirrer and condenser, a mixture of 187 mg (1.5 equiv.)
of triphenylphosphine (1.5 mmol/g) bounded on polymer (200e

736
G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
3.2.2. Antibodies
Kinase activities were assayed in Buffer D (þ0.5 mg BSA/
Anti-DYRK1A (H00001859-M01, 1:1000) and anti-GSK-3
(KAM-ST002E,1:1000) were obtained from Interchim and Stessgen,
respectively.
a/
b
mL þ 1 mM DTT, except for CDK2), at 30 ꢀC, at a final ATP concen-
tration of 15 mM. Blank values were subtracted and activities
expressed in % of the maximal activity, i.e. in the absence of in-
hibitors. Controls were performed with appropriate dilutions of
DMSO. The GS-1, CKS, CDK7/9 tide and RS peptide substrates were
obtained from Proteogenix (Oberhausbergen, France).
3.2.3. Affinity chromatography on immobilized Leucettines
3.2.3.1. Preparation of Leucettine agarose beads. CNBr-activated
sepharose 4B (Sigma) was swollen in cold 1 mM HCl for 30 min.
Beads were then activated with coupling buffer. Leucettine L41 or
analogue with a linker were synthesized as described above and
were coupled overnight under constant rotation at room tempera-
ture. After removal of the supernatant, residual active sites were
quenched using blocking buffer for 2 h 30 min under constant
rotation at room temperature. Beads were washed with coupling
buffer, washing buffer and bead buffer and brought to a 20% sus-
pension in bead buffer. They were stored at 4 ꢀC until further use.
Ethanolamine beads were used as controls.
3.2.5.2. CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin
E and CDK5/p25 (human, recombinant). CDK1/cyclin B (M phase
starfish oocytes, native), CDK2/cyclin E and CDK5/p25 (human,
recombinant) were prepared as previously described [15]. Their
kinase activity was assayed in buffer D (þ0.5 mg BSA /mL þ 1 mM
DTT, except for CDK2), with 1 mg histone H1/mL, in the presence of
15
30
m
m
M [
g-
33P] ATP (3000 Ci/mmol; 10 mCi/mL) in a final volume of
L. After 30 min incubation at 30 ꢀC, the reaction was stopped by
harvesting onto P81 phosphocellulose papers (Whatman) using
a FilterMate harvester (Packard) and was washed in 1% phosphoric
acid. Scintillation fluid was added and the radioactivity measured
in a Packard counter.
3.2.3.2. Preparation of brain extracts. Brains were obtained from
mice at the animal facility of the University of Rennes and snap-
frozen until further use. Tissues were weighed, homogenized and
sonicated in homogenization buffer (5 mL/g of material). Homog-
enates were centrifuged for 15 min at 17,000ꢂg at 4 ꢀC. The su-
pernatant was recovered, assayed for protein content (BioRad
Protein Assay) and kept at ꢁ80 ꢀC until use.
3.2.5.3. CDK9/cyclin T (human, recombinant, expressed in insect
cells). CDK9/cyclin T (human, recombinant, expressed in insect
cells) was assayed as described for CDK1/cyclin B, but using CDK7/9
tide (YSPTSPSYSPTSPSYSPTSPSKKKK) (8.1
mg/assay) as a substrate.
3.2.3.3. Preparation of extracts of other tissues. The different mice
tissues (brain, kidney, liver, lung, heart, stomach, intestines, colon,
muscle, spleen, ovary, thymus and skin) were obtained from the
University of Rennes animal facility and snap-frozen until further
use. Tissues were weighed, homogenized and sonicated in homog-
enization buffer (5 mL/g of material for all tissues except 10 mL/g for
kidney and liver). Homogenates were centrifuged for 15 min at
17,000ꢂg at 4 ꢀC. The supernatant was recovered, assayed for protein
content (BioRad Protein Assay) and kept at ꢁ80 ꢀC until use.
3.2.5.4. GSK-3 (porcine brain, native). GSK-3a/b
a
/
b
(porcine brain,
native) was assayed, as described for CDK1 but using a GSK-3
specific substrate (GS-1: YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS
stands for phosphorylated serine) [34].
3.2.5.5. CK1d/ε (porcine brain, native). CK1
d/ε (porcine brain,
native) was assayed as described for CDK1 but using 25
peptide (RRKHAAIGpSAYSITA), a CK1-specific substrate [35].
mM CKS
3.2.5.6. DYRK1A, 1B, 2 and 3 (Human, recombinant, expressed in
E. coli as GST fusion proteins) and CLK1, 2, 3 and 4 (mouse, recom-
binant, expressed in E. coli as GST fusion proteins). DYRK1A, 1B, 2
and 3 (Human, recombinant, expressed in E. coli as GST fusion
proteins) and CLK1, 2, 3 and 4 (mouse, recombinant, expressed in E.
coli as GST fusion proteins) were purified by affinity chromatog-
raphy on glutathione-agarose and assayed as described for CDK1/
3.2.3.4. Affinity chromatography of Leucettine L41-interacting pro-
teins. Just before use, 10 mL of packed beads were washed with 1 mL
of bead buffer and resuspended in this buffer. After a brief spin at
17,000ꢂg, the tissue extract supernatant (1 or 2 mg of total protein)
was loaded on 10 mL of packed beads and the volume was adjusted to
1 mL with bead buffer. The tubes were rotated at 4 ꢀC for 30 min.
After a brief spin at 10,000ꢂg and removal of the supernatant, the
cyclin
a substrate.
B with RS peptide (GRSRSRSRSRSR) (1 mg/assay) as
beads were washed 4 times with bead buffer before addition of 80 mL
of 2ꢂ LDS sample buffer (Invitrogen) and 200 mM DTT. Following
heat denaturation for 3 min, the bound proteins were analyzed by
SDS-PAGE and Western blotting or silver staining as described below.
4. Discussion
In this article we report on the synthesis and affinity chroma-
tography use of immobilized Leucettines. Leucettines constitute
a promising class of kinase inhibitors in various therapeutic areas,
with particular focus on Alzheimer’s disease and Down syndrome
at the moment [12,13]. It is therefore particularly important to
develop methods allowing the identification of the primary and
secondary targets of these molecules. Immobilization of Leu-
cettines through a polyethylene linker was chosen as one method
to analyze their selectivity since this approach has yielded a wealth
of information when applied to other kinase inhibitors [14e17]. In
addition as we had co-crystal structures of Leucettine L41 with
several kinases available [12,13], it was possible to rationally decide
where the linker should be attached to Leucettines without pre-
venting their interaction with kinase targets. This is in fact dem-
onstrated by the fact that Leucettine L41 without (1a) or with 4 EG
(3b) display essentially the same kinase inhibitory selectivity pro-
files and potency (Table 2). However this method excludes poten-
tial targets that might interact with Leucettines just at the site
3.2.4. Electrophoresis and Western blotting
The proteins bound to the matrices were separated by 10%
NuPAGE pre-cast BiseTris polyacrylamide mini gel electrophoresis
(Invitrogen) with MOPS-SDS running buffer followed by silver
staining (GE Healthcare Life Sciences, PlusOne Silver Staining Kit
Protein, 17-1150-01) or immunoblotting analysis. Proteins were
transferred to 0.45 mm nitrocellulose filters (Whatman). These were
blocked with 5% low fat milk in Tris-buffered saline/Tween 20 and
incubated overnight at 4 ꢀC with antibodies. Appropriate secondary
antibodies conjugated to horseradish peroxidase (BioRad) were
added to visualize the proteins using the Enhanced Chem-
iluminescence reaction (ECL, Amersham).
3.2.5. Protein kinase assays
3.2.5.1. Buffer D. MgCl2 (10 mM), 1 mM EGTA (ethyleneglycoltetra-
acetic acid), 1 mM DTT (dithiothreitol), 25 mM Tris/HCl, (50
heparin).
mg/mL

G. Burgy et al. / European Journal of Medicinal Chemistry 62 (2013) 728e737
737
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where the linker is attached. It would therefore be of interest to
immobilize Leucettines at some other sites as well, such as in the
methylenedioxy moiety. This might uncover yet undetected targets.
This possibility is supported by the fact that the kinase inactive
Leucettine 1b shows modest but detectable activity when a PEG
arm is attached (3c) (Table 2). In addition immobilized 3c binds
proteins including DYRK1A and GSK-3 (Fig. 2). These results suggest
some interaction directly with the linker itself or through physico-
chemical properties changes brought about in this area by the
linker attachment. Nevertheless the specificity in binding to Leu-
cettine beads was demonstrated by the fact that excess free ligand
added to the cellular or tissue extracts prior to and during binding
to immobilized Leucettine beads, resulted in reduced binding of the
targets [13]. Another drawback of the affinity chromatography
approach is the fact that it favors detection of the most abundant
targets. Nevertheless all the identified targets found by affinity
chromatography on immobilized Leucettines were also identified
by a competitive affinity method (based on binding competition
with immobilized broad spectrum kinase inhibitors which are able
to concentrate most of the expressed kinome from any cell or tissue
extract) [13]. Altogether, we believe we have established a solid
synthesis protocol to prepare Leucettines for immobilization onto
agarose beads, thereby providing a very useful reagent to purify,
concentrate and identify Leucettines’ targets from any biological
source. As an illustration we have shown here that the expression
pattern of Leucettine L41 in mouse tissues varies considerably from
one organ to the other. Our aim is to develop this method further
and to apply it to the most disease-relevant Leucettines with
appropriate target tissue and cell samples.
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This research was supported by grants from the ‘Fonds Unique
Interministériel” (FUI) PHARMASEA project (LM), the “Association
France-Alzheimer (Finistère)” (LM) and “Fondation Jérôme
Lejeune” (LM). We are thankful to Charlène BENESTEAU and Lau-
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for providing the mouse tissues. Guillaume BURGY is recipient of
a “CIFRE” PhD fellowship and Tania TAHTOUH of a PhD fellowship
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