Application of FITC-labeled ternatin on its cellular localization in 3T3-L1 murine preadipocytes

Article abstract of DOI:10.1246/cl.2009.150

The cellular localization of a potent fat-accumulation inhibitor (-)-ternatin was investigated. FITC-labeled ternatin, a chemical probe for current study, was synthesized from a highly bioactive ternatin analogue using Click Chemistry, which was found to

Full text of DOI:10.1246/cl.2009.150

150
Chemistry Letters Vol.38, No.2 (2009)
Application of FITC-labeled Ternatin on Its Cellular Localization
in 3T3-L1 Murine Preadipocytes
Kenichiro Shimokawa,1 Osamu Ohno,1 Kaoru Yamada,1 Yuichi Oba,2 and Daisuke UemuraÃ3
1Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602
2Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601
3Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522
(Received November 20, 2008; CL-081100; E-mail: uemura@bio.keio.ac.jp)
The cellular localization of a potent fat-accumulation in-
hibitor (À)-ternatin was investigated. FITC-labeled ternatin, a
chemical probe for current study, was synthesized from a highly
bioactive ternatin analogue using Click Chemistry, which was
found to be bioactive. The treatment of 3T3-L1 murine preadi-
pocytes with synthetic probe revealed that (À)-ternatin is local-
ized in a specific organelle of 3T3-L1 cells.
(À)-Ternatin (1, Figure 1) is a highly N-methylated cyclic
heptapeptide that was isolated from the mushroom Coriolus ver-
sicolor in our continuing search for potential antiobesity drugs
from natural sources.1 The in vitro biological evaluation re-
vealed that 1 potently inhibited fat-accumulation against 3T3-
L1 murine adipocytes. In addition, we also demonstrated the in-
hibitory effect of fat accumulation in vivo, which led to suppres-
sion of body-weight gain in high-fat-diet induced obese mice by
treatment with 1 (5 mg/kg/day) for 5 weeks.2 Therefore, 1 could
be considered as a plausible lead compound for antiobesity
drugs. However, both the biological mechanism of 1 and its
cellular target still remained unknown.
As an initial effort toward bioorganic studies on 1, we
recently reported its structure–activity relationships, which re-
vealed key amino acid residues (Ile1 and Leu4) responsible for
potent bioactivity.3 These SAR profiles enabled further chemical
modification, i.e., installation of functional groups, in appropri-
ate positions in the chemical structure of 1. In a previous paper,
we reported [NMe-D-ProGly6]-ternatin (2, Figure 1) as a highly
bioactive analogue of 1, which was applied to the synthesis of
biotin-labeled ternatin as a chemical probe for identification of
bio-molecules that bind to 1.4 As a part of our on-going bio-
organic studies on 1, we describe here the synthesis and biolog-
Scheme 1. a) 50% TFA/CH2Cl2; b) FITC, NEt3, DMF, 59% in
2 steps; c) 2 (1.0 equiv), CuSO4, sodium ascorbate, t-BuOH,
H2O, 58%; d) 50% TFA/CH2Cl2; e) FITC, NEt3, DMF, quant.
in 2 steps.
ical evaluation of FITC-labeled ternatin 3, and its cellular local-
ization in 3T3-L1 murine preadipocytes.
On the basis of the synthetic route to biotin-labeled com-
pounds4 which feature the Cu-catalysed Huisgen reaction (Click
Chemistry),5 we thought that 3 could be synthesized from three
components, 2, FITC (fluorescent isothiocyanate), and linker 4,
which is shown in Scheme 1. First, compound 4, prepared from
triethylene glycol in five steps, was subjected to Boc deprotec-
tion under acidic conditions. The resulting amine (as a TFA salt)
was coupled with FITC in the presence of NEt3 to provide the
FITC-linker conjugate 5. Finally, the Cu-catalyzed Huisgen
reaction of 5 with alkyne 2 using CuSO4 and sodium ascorbate
in t-BuOH/H2O generated 3. After HPLC purification of the
reaction mixture, the desired 3 was obtained in 58% yield. On
the other hand, another FITC-linker conjugate 7 which bears a
methyl ester group instead of the peptide core in 3 was synthe-
sized as a negative compound in our study. The Boc deprotection
of 6 followed by coupling with FITC afforded 7.
Figure 1. Structures of 1 and 2.
Copyright ꢀ 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.2 (2009)
151
Table 1. Fat-accumulation inhibitory effects of compounds 1–3
and 7, and cell viability of 3T3-L1 adipocytesa
Fat-acumulation
inhibitory effect: IC50
Cell viability: IC50
/mM
Compound
/mM
(À)-Ternatin (1)
0:027 Æ 0:003
0:019 Æ 0:001
23 Æ 1:5
0:28 Æ 0:03
>5:2b
2
3
7
>75b
>164b
>164b
aValues are means of quadruplicate determinations. bNot tested
at higher concentrations.
The in vitro fat-accumulation inhibitory effects for synthetic
compounds 3 and 7 were then assessed (Table 1). The bioassay
consisted of the treatment of confluent 3T3-L1 preadipocytes
with each sample and insulin (an inducer of adipogenesis), and
further incubation for 7 days. After this period, control cells were
differentiated into mature adipocytes. Both the rates of fat accu-
mulation and cell viability were calculated to identify undesired
fat-accumulation inhibition caused by cytotoxicity.
On the basis of the results, 3 showed moderate inhibitory
effect on fat accumulation with an IC50 value 23 mM and no sig-
nificant cytotoxicity at tested concentrations (IC50 > 75 mM),
which suggested that 3 was potent enough to be applied for fur-
ther bio-organic investigation. Meanwhile, 7 did not show any
inhibitory effect or cytotoxicity (IC50 > 164 mM).
Visualization of the intracellular localization of synthesized
compounds was conducted using fluorescence microscopy
(Eclipse TE200; Nikon, Tokyo, Japan), which is shown in
Figure 2.6 For this examination, we employed 3T3-L1 murine
preadipocytes. Our recent research demonstrated that 1 shows
its inhibitory effects against fat-accumulation by suppressing
the differentiation of 3T3-L1 cells in the early stage. Therefore,
1 was thought to show its biological effects in 3T3-L1 murine
preadipocytes.
By the fluorescence microscopic analysis, a certain rate of
3T3-L1 cells treated with 3 showed strong fluorescence under
UV (Figure 2b). The pattern of the fluorescence was partial
and spot-like inside the cells. On the other hand, no fluorescence
was observed when cells were treated with 7 (Figure 2d). These
results suggested that 3 was incorporated into cells and localized
in a specific organelle of 3T3-L1 cells. It was confirmed that the
organelle where 3 accumulated was not identical to nucleus by
3 and DAPI co-staining analysis (data not shown). The part of
localization is now under investigation. Thus, FITC-labeled
(À)-ternatin was demonstrated to localize in a specific organelle
of 3T3-L1 cells, and then, (À)-ternatin is likely to function there
to show its biological activity.
In summary, we synthesized FITC-conjugated ternatin 3 us-
ing Click Chemistry, and examined its inhibitory effect against
the fat accumulation in 3T3-L1 cells. Next, we surveyed its in-
tracellular localization in 3T3-L1 cells using fluorescence micro-
scopic analysis. 3 was observed to localize in a specific organelle
of 3T3-L1 cells, and thus, 3 will be a useful tool for the analysis
of the mechanism of action and the identification of the target
molecules of 1.
Figure 2. Cellular localization of FITC-labeled ternatin 3 in
3T3-L1 murine preadipocytes analyzed by fluorescence. (a)
Cells treated with 3 for 18 h; (b) fluorescence image of a;
(c) cells treated with 7 for 18 h; (d) fluorescence image of c;
(e) control cells (no additive); (f) fluorescence image of e.
We thank Prof. M. Ojika (Nagoya University) for kind per-
mission to use fluorescence microscopy. This study was support-
ed in part by Grants-in-Aid for Scientific Research for Creative
Scientific Research (Grant No. 16GS0206) and the Global COE
program in Chemistry at Nagoya University (Grant No. B-021),
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan. We are indebted Banyu Pharmaceutical
Co., Ltd. for their financial support.
References and Notes
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6
3T3-L1 preadipocytes were inoculated at 5 Â 104 cells/well in 24-well
plates, and incubated with 25 mM of 3 or 7 for 18 h. After washing, cells
were analyzed by phase contrast (left panels) and fluorescence (right
panels) microscopy. The cells were photographed at a magnification
of 200Â.
Published on the web (Advance View) January 10, 2009; doi:10.1246/cl.2009.150