D.-m. Tian et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 1. Representative structure of cardiac glycoside probes.
of endothelin-1 (ET-1) expression in vascular endothelial cells (ECs)
through an inhibitory interaction with the c-Jun/AP-1 pathway. Yan
cardiac glycosides from the plant of Antiaris toxicaria, were selected to
prepare the chemical probes due to their significant difference in in-
duction Nur77 expression bioactivities (β-Antiarin is better than α-
Antiarin). A linker between the bioactive compound and the tag
(biotin) should be flexible and have appropriate length in order to
8
et al. reported that Nur77 is a novel negative regulator for the β-
adrenergic receptor (β-AR)-induced cardiac hypertrophy through in-
hibiting the NFATc3 and GATA4 transcriptional pathways. To sum up,
Nur77 mediates many important physiological and pathological pro-
cesses of humankind diseases.
2
1
provide enough space for biotin to interact with streptavidin. Mean-
while, the amphiphilicity of the linkers influences the penetrability, and
2
1
Cardiac glycosides have been used for the treatment of congestive
heart failure (CHF) for centuries and their known cellular target is the
then the bioactivity of the chemical probes. Initially, the hydrophobic
2
diamine [NH –(CH
2
)n-NH ] and hydrophilic PEG-bis(amine) with dif-
2
+
+
12,13
alpha subunit of the sodium (Na )/potassium (K )-ATPase.
Our
ferent chain length were used as the linkers. We proposed that one
primary amine of the linker reacted with the carboxylic acid of the
biotin to form amide bond and the other primary amine of the linker
reacted with formyl group of C-10 of β-Antiarin (α-Antiarin) to form
imine bond. However, the products were obtained in low yields and
were unstable due to the instability of imine bond. Then, an alternative
synthetic route was designed by introduction of hydrazine between
linkers and the formyl group (–CHO) of cardiac glycosides. Two kinds
previous studies showed that treatment of cancer cells with cardiac
glycosides significantly resulted in the expression of Nur77 protein at a
concentration of 20 nM. Immunofluorescence assay displayed that the
induced Nur77 protein subsequently translocated from the nucleus to
the cytoplasm and subsequent targeting to mitochondria, leading to
+
+
apoptosis of cancer cells, which may be independent on Na /K -AT-
1
4,15
Pase.
However, it’s still unclear that how does cardiac glycosides
affect the Nur77 signaling pathway.
of linker, [NH
2
–(CH
2
)n-COOH and NH
2
–(CH
2
–CH
2
–O)n-CH –COOH],
2
Compound-centric chemical proteomics (CCCP) is a very powerful
technique to unveil the specific molecular targets of bioactive com-
were introduced to formyl group at C-10 of β-Antiarin (α-Antiarin) after
reacting with hydrazine (Fig. 1). The synthetic probes were obtained in
high yield and were stable under neutral conditions. To our knowledge,
this is the first time for systematic synthesis of biotinylated cardiac
glycosides for fishing their target proteins.
1
6
pounds, which has been successfully applied for identifying the cel-
1
7
18
19
lular targets of NSC751382, Orlistat, β-lactones and a variety of
2
0
natural products.
CCCP combines the affinity chromatography
method and advanced mass spectrometry technique whereby the small
molecule compound is covalently immobilized to a solid support, and
incubated with a protein lysate to pull down the interacting proteins
which will be identified using LC–MS/MS subsequently. Biotin is an
optimal tag for labeling the bioactive molecules due to its strongest
non-covalent biological interaction with streptavidin, which made it
feasible for the enrichment and purification of biomolecule(s) binding
to the biotinylated chemical probes. Here, in order to probe the Nur77
protein inducting pathway, we designed and synthesized a series of
novel biotinylated cardiac glycosides from β-Antiarin and α-Antiarin
for fishing the molecular target (s) binding to cardiac glycosides.
Design of biotinylated cardiac glycosides for probing Nur77 protein in-
ducting pathway. Generally, chemical probe is consisted of three parts
including a tag easy to detect or to purify, a flexible linker, and the
Synthesis of biotinylated cardiac glycosides P1a and P1b from
Biotinylhydrazine (B-L-1) and β-Antiarin (α-Antiarin). The synthesis of
biotinylated cardiac glycosides P1a and P1b started from the synthesis
of Biotinylhydrazine (B-L-1) which was prepared according the litera-
2
3
ture. With the biotinylhydrazine (B-L-1) and cardiac glycosides (β-
Antiarin and α-Antiarin) in hands, probes P1a and P1b were synthe-
2
4
sized by the hydrazone formation under the catalysis of 1% AcOH.
The crude products were obtained by concentrated under reduced
pressure. The residues were purified by semipreparative RP-HPLC (35%
MeOH-H O) to obtain P1a (50.8% yield) and P1b (41.8% yield)
2
(Scheme 1). The products P1a and P1b were both identified as mixtures
of trans and cis epimers due to the presence of imine bond at C-19. The
ratios of the trans and cis epimers of P1a and P1b were both about 3:1,
1
which were identified by the analysis of H NMR spectra.
2
1
bioactive compound that reacts with the target(s). Here, biotin was
chosen as the tag. The selection of the bioactive compound determines
the efficiency of the chemical probes. What’s more, it is better that
biotinylation of the bioactive compound has no or less effect on its
bioactivity. Structure-activity relationship (SAR) studies demonstrated
that the sugar moiety in C-3 of cardiac glycosides is necessary for its
induction of Nur77 expression. Meanwhile, the substituents at C-10 of
cardiac glycosides showed significant effects on their bioactivities, with
Synthesis of biotinylated cardiac glycosides P2a-P5a and P2b-P5b from
Biotin–AA
n
–CONHNH
2
2
(B-L-2–B-L-5) and β-Antiarin (α-Antiarin).
(n = 1, 3, 5, and 7, B-L-2–B-L-5) were prepared
Biotin–AA
n
–CONHNH
firstly. Biotin was activated by N-hydroxysuccinimide (NHS) in the
presence of N,N′-dicyclohexylcarbodiimide (DCC) to obtain Biotin–NHS
2
5
as reported methods. Then, Biotin–NHS was reacted with hydro-
phobic linkers [glycine, γ-aminobutyric acid, 6-aminocaproic acid and
8-aminooctanoic acid, AA (n = 1, 3, 5 and 7)] to achieve biotin-linker
25,26
n
methyl (–CH
3
), hydroxymethyl (–CH
2
OH), or formyl group (–CHO)
products (Biotin–AA
(n = 1, 3, 5, and 7, B-L-2–B-L-5) were prepared by the reaction of B-
AAn-COOH with SOCl under excess MeOH and subsequent hy-
drazinolysis with hydrazine hydrate in MeOH.
The prepared Biotin–AA –CONHNH (B-L-2–B-L-5) were reacted
with β-Antiarin and α-Antiarin under the catalysis of 1% AcOH in
n
–COOH).
Finally, Biotin–AA
n
–CONHNH
2
substituents at C-10 exhibiting comparable effects on induction of
1
4,15,22
Nur77 expression.
Based on the SAR analysis and the relatively
2
2
3
conservative structures of cardiac glycosides, we decided to biotinylate
cardiac glycosides at the formyl group (–CHO) of C-10, which is highly
reactive chemically. Finally, β-Antiarin and α-Antiarin, two typical
n
2
2