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-1-
Total Synthesis of a New Fluorescent Probe for
the Application to Intracellular Calcium Ions
He Huaizhen, Lei Lei, Cheng Zhao, Li Jianli, Shi Zhen *
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of
Education, Department of Chemistry, Northwest University，Xi’an（710069）
E-mail: selenaorganic@163.com
Abstract
In this study, a new kind of calcium-selective fluorescent probe Fluo-3ClMM-OH was synthesized.
The structure of reaction products were characterized by IR and
1
H NMR spectra. The molecular
weights were measured on MALDI-TOF MS. Methylation of the crude product provided an
effective method for separation. The fluorescence emission wavelength maxima of
Fluo-3ClMM-OH were also researched.
Keywords: Calcium ions; fluorescent probe; methylation; fluorescence emission
1. Introduction
Calcium plays a critical role in a broad variety of biological systems. Numerous functions
of all types of cells are regulated by Ca
2+
to a greater or lesser degree,
1,2
such as calcium’s role
in the beginning of life and the act of fertilization. Furthermore, it was shown that Ca
2+
acted as
a universal second messenger and Ca
2+
-binding proteins existed in a variety of cells, which
facilitated analysis of the potential functions of Ca
2+
in a variety of cells.
3-5
Because of the
importance of Ca
2+
in biology, numerous techniques and methods for investigating the
mechanisms of cellular and subcellular Ca
2+
activity have been developed. Among numerous
methods, those based on fluorescence enjoy the benefits of high sensitivity, convenience of
dynamic studies and even provide the possibility to visualize the Ca
2+
distribution inside a
cell.
6
They are highly sensitive and offer imaging by fluorescent microscopy in an easier and
less cell damaging way than other methods (e.g., ion-selective electrodes, NMR
techniques).
7-10
Despite of intensive research efforts, only a few fluorescent indicators are
commercially available.
11,12
Among them Fluo-3 is a visible-wavelength calcium ions
indicator which is convenient for confocal laser scanning microscope (CLSM) and flow
cytometry.
13
However, Fluo-3 also suffers some drawbacks.
14
Its somewhat low affinity to Ca
2+
does not allow Fluo-3 to apply for the observation of small variations in Ca
2+
concentration.
The fluorescence signal is also not satisfactory.
Here we report the design and synthesis of an improved visible-wavelength
calcium-selective fluorescent probe Fluo-3MM-OH. It has in common the principle of
covalently linking an organic fluorophore to a Ca
2+
-chelating moiety, mostly to 1,2 - bis -
( 2-amino-phenoxy ) ethane - N, N, N’, N’ tetraacetic acid (BAPTA). Introduction of three
methyl groups into the Ca
2+
-specific binding site of BAPTA may increase the electron density
and thus improves the affinity of the probe towards a calcium ion. The synthetic route of the
compounds is mentioned in Scheme 1. Methylation of the crude Fluo-3ClMM-OH, which
reduced the polarity of the product provided an effective method for separation.
NO
2
O
O
NO
2
CH
3
H
3
C
NO
2
O
H
3
C
NO
2
OH
H
3
C
a
b
c
Cl
Cl
CH
3
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