Synthesis of new BAPTA calcium chelators

Article abstract of DOI:10.1080/00397910802633421

A series of new 1,2-bis-(2-aminophenoxyethane) N,N,N',N'-tetraacetic acid (BAPTA) chelators were designed and synthesized to form novel and improved fluorescent indicators. Substituted 2-nitrophenol, acting as the material, reacted in five steps to give BAPTA-derived chelators. The Ca2+ affinity of BAPTA chelators could increase significantly by addition of electron-donating groups and decrease by electron-withdrawing groups. Compounds 3a-e were treated with iron powder instead of hydrogenation, obtaining a satisfying yield. The structures of all compounds were characterized by infrared, 1H NMR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra.

Full text of DOI:10.1080/00397910802633421

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Synthesis of New BAPTA
Calcium Chelators
Huai Zhen He a , Lei Lei a , Jian Li Li a & Zhen Shi a
a Key Laboratory of Synthetic and Natural
Functional Molecular Chemistry, Education Ministry ,
Department of Chemistry, Northwest University ,
Xi'an, China
Published online: 13 May 2009.
To cite this article: Huai Zhen He , Lei Lei , Jian Li Li & Zhen Shi (2009) Synthesis of
New BAPTA Calcium Chelators, Synthetic Communications: An International Journal
for Rapid Communication of Synthetic Organic Chemistry, 39:12, 2074-2081, DOI:
10.1080/00397910802633421
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Synthetic Communications1, 39: 2074–2081, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910802633421
Synthesis of New BAPTA Calcium Chelators
Huai Zhen He, Lei Lei, Jian Li Li, and Zhen Shi
Key Laboratory of Synthetic and Natural Functional Molecular
Chemistry, Education Ministry, Department of Chemistry,
Northwest University, Xi’an, China
Abstract: A series of new 1,2-bis-(2-aminophenoxyethane) N,N,N0,N0-tetraacetic
acid (BAPTA) chelators were designed and synthesized to form novel and improved
fluorescent indicators. Substituted 2-nitrophenol, acting as the material, reacted in
five steps to give BAPTA-derived chelators. The Ca2þ affinity of BAPTA chelators
could increase significantly by addition of electron-donating groups and decrease by
electron-withdrawing groups. Compounds 3a–e were treated with iron powder
instead of hydrogenation, obtaining a satisfying yield. The structures of all
1
compounds were characterized by infrared, H NMR, and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra.
Keywords: BAPTA, chelator, ion indicator, synthesis
INTRODUCTION
Calcium plays a critical role in a broad variety of biological systems.
Numerous functions of all types of cells are regulated by Ca2þ to a
greater or lesser degree, such as calcium’s role in the beginning of life
and the act of fertilization.[1–4] Furthermore, it has been shown that
Ca2þ acts as a universal second messenger and Ca2þ-binding proteins
exist in a variety of cells, which facilitate analysis of the potential
functions of Ca2þ in a variety of cells.[5–7] Because of the importance of
Received September 7, 2008.
Address correspondence to Huai Zhen He, Key Laboratory of Synthetic and
Natural Functional Molecular Chemistry, Education Ministry, Department of
Chemistry, Northwest University, Xi’an 710069, China. E-mail: selenaorganic@
163.com
2074

Synthesis of New BAPTA Calcium Chelators
2075
Ca2þ in biology, measuring the temporal and spatial variations of the
intracellular free calcium concentration ([Ca2þ]i) becomes a common
theme in cytometry, cell biology, and neuroscience. Among numerous
methods for investigating the [Ca2þ]i, those based on fluorescence enjoy
the benefits of good sensitivity and convenience of dynamic studies and
even provide the possibility of visualizing the Ca2þ distribution inside a
cell.[8] They are very sensitive and offer imaging by fluorescent micro-
scopy in an easier and less-cell-damaging way than other methods, such
as ion-selective electrodes and NMR techniques.[9–13]
These fluorescent indicators for metal ions have in common the
principle of covalently combining an organic fluorophore with a Ca2þ
chelating moiety to give rhodamine-like or fluorescein-like indicators.[14]
Virtually all of the chelator compounds used for these applications have
been derivatives of iminodiacetic acid or related amine aliphatic com-
pounds, such as ethylenediamine tetraacetic (EDTA), diethylenetriamine-
pentaacetic (DTPA), or ethyleneglycolbis (2-aminoethylether)-N,N,N0,N0-
tetraacetic acid (EGTA). Among these, the most selective ones for
Ca2þ are derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N0,N0-
tetraacetic acid (BAPTA). The most widely used Ca2þ indicators, such
as quin-2, fura-2, indo-1, fluo-3, rhod-2, and fluo-4, have incorporated
this basic BAPTA structure.[15] The Ca2þ affinity of BAPTA chelators
is about the same at pH 7 as it is at greater values. In addition, the affinity
of BAPTA-derived chelators can be significantly changed by the addition
of different groups: increased by addition of electron-donating groups
and decreased by electron-withdrawing groups.[16] Therefore, there is still
an urgent need to develop an effective and practical method to synthesize
new BAPTA chelators.
In this article, we report the design and synthesis of a series of new
BAPTA-derived chelators by addition of different groups (Fig. 1). There
into, R is selected from the group consisting of NH2, Li, Br, or CHO,
which can act as functional groups to form a covalent bond between
the chelators and the fluorophore. Here, we choose aldehyde as the func-
tional group. The incorporation of such nonfunctional groups as R1, R2,
and R3, which are selected from H, CH3, Cl, can be used to change the
Figure 1. Structure of the new BAPTA-derived chelators.

2076
H. Z. He et al.
Scheme 1. Synthetic route of the new BAPTA-derived chelators.
affinity of Ca2þ. We can make use of this property to design novel
and improved fluorescent indicators for measuring the intracellular free
calcium concentration. The synthetic route of the new BAPTA-derived
chelators is shown in Scheme 1. Iron powder was used as the reducing
agent instead of hydrogenation, obtaining a satisfying yield.[17]
EXPERIMENTAL
Starting materials were commercially available unless otherwise noted.
All reagents employed were purchased from Xi’an Chemical Reagent
Corp. The melting points were measured on an XT-4 instrument without
1
correction. H NMR spectra were recorded at 400 MHz in CDCl3 with
tetramethylsilane (TMS) as an internal reference. The infrared (IR) spec-
tra were recorded on a Shimadzu Fourier transform (FT)–IR 440 spectro-
meter in the 4000–500 cmꢀ1 range. The molecular weights were measured
on a matrix-assisted laser desorption=ionization time-of-flight mass spec-
trometry (MALDI-TOF MS), using a-cyano-4-hydroxycinnamic acid
(CHCA) as a reference frame. The synthetic procedure was controlled
by the method of thin-layer chromatography (TLC). The products were
purified by recrystallization.
1-(2-Chloro-ethoxy)-4-methyl-2-nitro-benzene (1a)
2-Nitro-phenol (1.53g,0.01 mol), 1-bromo-2-chloroethane(4.30g,0.03 mol),
and K2CO3 (2.07 g, 0.015 mol) were dissolved in dimethylformamide (DMF)

Synthesis of New BAPTA Calcium Chelators
2077
(8 mL). The reaction mixture was stirred at 120ꢁC for 5 h. The solution
color changed from dark orange to yellow. After the reaction, the mix-
ture was cooled, diluted with EtOAc (20 mL), and then washed with
water (3 ꢂ 10 mL). The organic layer was collected and concentrated
under strong vacuum. The residue was dissolved in MeOH (5 mL). The
MeOH solution was cooled overnight to crystallize. The light yellow solid
1
was collected by filtration. Yield 96%; mp 33–34ꢁC; H NMR (CDCl3,
400 MHz) d 7.657 (s, 1H), 7.339 (d, J ¼ 8.8 Hz, 1H), 6.993 (d, J ¼ 8.8 Hz,
1H), 4.388 (t, J ¼ 6.0 Hz, 1H), 4.332 (t, J ¼ 6.0 Hz, 1H), 3.848 (t, J ¼
6.0 Hz, 1H), 3.665 (t, J ¼ 6.0 Hz, 1H), 2.360 (s, 3H); IR (neat) 3420.9,
2930.3, 1626.9, 1530.3, 1357.4, 1258.1, 1157.8, 1030.6, 810.7 cmꢀ1; TLC
Rf ¼ 0.70 in 2:1 hexanes=EtOAc.
4-Methyl-2-(2-(4-methyl-2-nitrophenoxy)ethoxy)-1-nitrobenzene (2a)
A mixture of 1a (1.94 g, 0.01 mol) and 5-methyl-2-nitro-phenol (1.65 g,
0.01 mol) were dissolved in DMF (10 mL). K2CO3 (2.50 g, 0.018 mol)
was added to the reaction mixture. The reaction mixture was stirred at
140–160ꢁC for 4–5 h. The solution color changed from dark orange to
light orange. After the reaction, the mixture was cooled, stood for 2 h,
and then poured into cold water (20 mL). The formed yellow solid was
collected by filtration and washed with water (3 ꢂ 5 mL) until the water
1
filtrate became colorless. Yield 94%; mp 148–150ꢁC; H NMR (CDCl3,
400 MHz) d 7.785 (d, J ¼ 8.8 Hz, 1H), 7.640 (s, 1H), 7.370 (d, J ¼ 8.8 Hz,
1H), 7.145 (d, J ¼ 8.8 Hz, 1H), 7.027 (s, 1H), 6.871 (d, J ¼ 8.8 Hz, 1H),
4.497 (s, 4H), 2.443 (s, 3H), 2.355 (s, 3H); IR (neat) 3427.8, 2930.9,
1602.9, 1521.1, 1349.1, 1253.5, 1172.9, 1057.9, 814.6 cmꢀ1; TLC
Rf ¼ 0.47 in 2:1 hexanes=EtOAc.
2-(2-(2-Amino-5-methylphenoxy)ethoxy)-5-methylbenzenamine (3a)
Iron powder (3.36 g, 0.06 mol), concentrated hydrochloric acid (0.2 mL),
and anhydrous ethanol (10 mL) were added into a dried three-
necked flask equipped with a magnetic stirrer. When the mixture was
heated to boiling, 2a (3.32 g, 0.01 mol) was added. The mixture was
refluxed for 4 h. At the end of the reaction, the hot mixture was just made
alkaline to litmus by addition of the calculated amount of 15% alcoholic
potassium hydroxide solution. Without the mixture becoming cool, the
iron powder was removed by filtration, and the reaction flask was rinsed
with 95% ethanol (2 ꢂ 5 mL). The formed white precipitate was obtained
by adding 6 mol=L sulfuric acid into the filtrate. Then the mixture was

2078
H. Z. He et al.
cooled to 25ꢁC and filtered. After that, the precipitate was dissolved in
40 mL of boiling water. When the solution was cooled to 40ꢁC it was
made alkaline with saturated sodium hydroxide solution. The formed
light yellow solid was collected by filtration. Yield 92%; mp 118–
1
120ꢁC; H NMR (CDCl3, 400 MHz) d 6.790 (d, J ¼ 8.0 Hz, 1H), 6.750
(d, J ¼ 8.0 Hz, 1H), 6.714 (s, 1H), 6.687 (s, 1H), 6.660 (d, J ¼ 8.0 Hz,
1H), 6.640 (d, J ¼ 8.0 Hz, 1H), 4.351 (s, 4H), 2.255 (s, 3H), 2.355
(s, 3H); IR (neat) 3440.9, 2926.2, 1624.7, 1517.9, 1227.2, 1157.3, 801.1,
574.6 cmꢀ1; TLC Rf ¼ 0.75 in 1:2 hexanes=EtOAc.
[(2-{2-[2-(Bis-methoxycarbonylmethyl-amino)-5-methyl-phenoxy]-
ethoxy}-5-methyl-phenyl)-methoxycarbonylmethyl-amino]-Acetic
Acid Methyl Ester (4a)
Compound 3a (2.72 g, 0.01 mol) was dissolved in MeCN (10 mL).
(i-Pr)2NEt (6 mL) and methyl bromoacetate (3 mL) were added to the
mixture with stirring. The reaction mixture was refluxed for 24–36 h.
After the reaction, the mixture was allowed to cool to 40–60ꢁC, poured
into ethyl acetate (20 mL), and then filtered to remove the formed white
solid. The combined ethyl acetate filtrates were concentrated in vacuo to
give an oily solid. A little methanol was added to the solid. Then, the sus-
pension was filtered to collect the formed white solid, which was further
1
washed with cold methanol and air dried. Yield 80%; mp 93–95ꢁC; H
NMR (CDCl3, 400 MHz) d 8.024 (s, 1H), 6.803 (d, J ¼ 8.8 Hz, 1H),
6.763 (s, 1H), 6.732 (d, J ¼ 8.8 Hz, 1H), 6.656 (d, J ¼ 9.6 Hz, 1H), 6.626
(d, J ¼ 9.6 Hz, 1H), 4.381 (s, 2H), 4.354 (s, 2H), 4.144 (s, 4H), 4.044
(s, 4H), 3.572 (s, 6H), 3.562 (s, 6H), 2.331 (s, 3H), 2.265 (s, 3H); IR (neat)
2953.9, 1751.3, 1518.9, 1418.2, 1255.1, 1170.7, 1004.5, 795.8, 710.9 cmꢀ1
;
TLC Rf ¼ 0.79 in 1:2 hexanes=EtOAc.
[(2-{2-[2-(Bis-methoxycarbonylmethyl-amino)-5-methyl-phenoxy]-
ethoxy}-4-formyl-5-methyl-phenyl)-methoxycarbonylmethyl-amino]-
Acetic Acid Methyl Ester (5a)
POCl3 (9.6 mL) was added dropwise into dry DMF (20 mL) over
40–45 min in a dry three-necked flask. The solution was stirred at rt for
1–2 h and then cooled. Compound 4a (5.60 g, 0.01 mol) was dissolved
in DMF (20 mL) and added dropwise into the POCl3=DMF mixture.
The reaction mixture was heated at 40–45ꢁC overnight. After the reac-
tion, the mixture was allowed to cool to rt, concentrated in vacuo, and
then poured into ice water. The suspension was filtered to collect the
1
solid. Yield 72%; mp 108–110ꢁC; H NMR (CDCl3, 400 MHz) d 10.135

Synthesis of New BAPTA Calcium Chelators
2079
(s, 1H), 7.315 (s, 1H), 6.790 (d, J ¼ 8.0 Hz, 1H), 6.699 (s, 1H), 6.673 (d,
J ¼ 8.0 Hz, 1H), 6.445 (s, 1H), 4.303–4.290 (m, 4H), 4.256–4.229
(m, 4H), 4.146 (s, 4H), 3.616 (s, 12H), 2.558 (s, 3H), 2.272 (s, 3H); IR
(neat) 3471.6, 2952.2, 2923.2, 1749.4, 1681.5, 1520.5, 1257.9, 1201.5,
1167.6, 1010.5, 711.4 cmꢀ1
; MALDI=TOF MS (m=z) calcd. for
C29H36N2O11 588.60; found 588.84; TLC Rf ¼ 0.59 in 1:2 hexanes=EtOAc.
Compounds 5b–e were obtained following the same procedure.
1
Compound 5b: Yield 78%; mp 150–152ꢁC; H NMR (CDCl3, 400 MHz)
d 10.130 (s, 1H), 7.305 (s, 1H), 6.807 (s, 1H), 6.685 (s, 1H), 6.451 (s, 1H),
4.272–4.268 (m, 4H), 4.261–4.220 (m, 4H), 4.104 (s, 4H), 3.609 (s, 12H),
2.553 (s, 3H), 2.273 (s, 3H); IR (neat) 3444.6, 2952.2, 2923.2, 1744.2,
1679.4, 1514.8, 1252.8, 1204.9, 1172.2, 1005.5, 709.1 cmꢀ1; MALDI=TOF
MS (m=z) calcd. for C29H35ClN2O11 623.05; found 623.24; TLC Rf ¼ 0.69
in 1:2 hexanes=EtOAc.
Compound 5c: Yield 81%; mp 107–109ꢁC; 1H NMR (CDCl3,
400 MHz) d 9.800 (s, 1H), 7.385 (d, J ¼ 8.0 Hz, 1H), 6.802 (s, 1H),
6.762 (d, J ¼ 8.0 Hz, 1H), 6.686 (s, 1H), 4.314–4.306 (m, 4H), 4.298–
4.295 (m, 4H), 4.230–4.104 (m, 4H), 3.630–3.562 (m, 12H), 2.278
(s, 3H); IR (neat) 3451.4, 2951.3, 1747.2, 1681.9, 1514.5, 1250.5,
1204.9, 1170.7, 1006.3, 713.4 cmꢀ1; MALDI=TOF MS (m=z) calcd for
C28H33ClN2O11 609.02; found 609.78; TLC Rf ¼ 0.66 in 1:2 hexanes=
EtOAc.
Compound 5d: Yield 72%; mp 130–131ꢁC; 1H NMR (CDCl3,
400 MHz) d 10.150 (s, 1H), 7.367 (s, 1H), 6.622 (s, 1H), 6.474 (s, 1H),
4.391–4.369 (m, 4H), 4.259–4.211 (m, 4H), 3.860–3.842 (d, 4H), 3.675–
3.625 (d, 12H), 2.564 (s, 3H), 2.521 (s, 3H), 2.336 (s, 3H); IR (neat)
2952.4, 2876.1, 1752.8, 1670.8, 1594.6, 1519.5, 1259.2, 1200.2, 1006.3,
708.2 cmꢀ1; MALDI=TOF MS (m=z) calcd. for C30H37ClN2O11 637.07;
found 636.35; TLC Rf ¼ 0.70 in 1:2 hexanes=EtOAc.
Compound 5e: Yield 77%; mp 103–105ꢁC; 1H NMR (CDCl3,
400 MHz) d 9.814 (s, 1H), 7.438 (s, 1H), 7.425 (d, J ¼ 8.0 Hz, 1H),
6.783 (d, J ¼ 8.0 Hz, 1H), 6.630 (s, 1H), 4.427–4.404 (t, 2H), 4.262–
4.239 (t, 2H), 4.220 (s, 4H), 3.642–3.602 (d, 12H), 2.525 (s, 3H), 2.339
(s, 3H); IR (neat) 3476.9, 2953.1, 1748.2, 1687.9, 1519.5, 1259.5,
1203.9, 1168.5, 1005.1, 982.5 cmꢀ1; MALDI=TOF MS (m=z) calcd. for
C29H35ClN2O11 623.05; found 622.50; TLC Rf ¼ 0.69 in 1:2 hexanes=
EtOAc.
In conclusion, we have designed and synthesized five derivatives of
BAPTA. The affinity of these BAPTA-derived chelators can be signifi-
cantly increased by the addition of electron-donating groups such as
methyl and decreased by electron-withdrawing groups such as chlorine.

2080
H. Z. He et al.
We can make use of this property to design novel and improved fluores-
cent indicators for measuring intracellular free calcium concentration.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of
China (20872117), the Ph.D. Program Foundation of Chinese University
(20050697003), and the Science and Technology Program Foundation of
Xi’ an City (YF07070).
REFERENCES
1. Tsien, R. Y.; Rev, A. Intracellular measurements of ion activities. Biophys.
Bioeng. 1983, 12, 101–106.
2. Dang, F. F.; Lei, K. W.; Liu, W. S. A new highly selective fluorescent silver
probe. J. Fluores. 2008, 18, 149–153.
3. Berridge, M. J. Inositol triphosphate and calcium signalling. Nature 1993,
361, 315–325.
4. Stricker, S. A.; Centonze, V. E.; Paddock, S. W.; Schatten, G. Confocal
microscopy of fertilization-induced calcium dynamics in sea urchin
eggs. Dev. Biol. 1992, 149, 370–380.
5. Whitaker, M.; Patel, R. Calcium and cell cycle control. Development 1990,
108, 525–542.
6. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals,
6th ed.; Molecular Probes, Eugene, OR, 2002.
7. Ridgeway, E. B.; Ashley, C. C. Calcium transients in single muscle fibers.
Biophys. Res. Commun. 1967, 29, 229–234.
8. Grynkiewicz, G.; Poenie, G. M.; Tsien, R. Y. A new generation of Ca2þ
indicators with greatly improved fluorescence properties. J. Biol. Chem.
1985, 260, 3440–3450.
9. Tsien, R. Y. A non-disruptive technique for loading calcium buffers and
indicator into cells. Nature 1981, 290, 527–528.
10. Tsien, R. Y.; Pozzan, T.; Rink, T. J. Calcium homeostasis in intact lympho-
cytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped
fluorescent indicator. J. Cell. Biol. 1982, 94, 325–334.
11. Komatsu, H.; Iwasawa, N.; Citterio, D.; Suzuki, Y.; Kubota, T. Design and
synthesis of highly sensitive and selective fluorescein-derived magnesium
fluorescent probes and application to intracellular 3D Mg2þ imaging.
J. Am. Chem. Soc. 2004, 126, 16353–16360.
12. Minta, A.; Kao, J. P.; Tsien, R. Y. Fluorescent indicators for cytosolic
calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem.
1989, 264, 8171–8178.
13. Harkins, A. B.; Kurebayashi, N.; Baylor, S. M. Resting myoplasmic free
calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys. J.
1993, 65, 865–881.

Synthesis of New BAPTA Calcium Chelators
2081
14. Gaillard, S.; Yakovlev, A.; Luccardini, C.; Oheim, M.; Feltz, A.; Mallet, J. M.
Synthesis and characterization of a new red-emitting Ca2þ indicator, calcium
ruby. Org. Lett. 2007, 9, 2629–2632.
15. Tsien, R. Y. New calcium indicators and buffers with high selectivity against
magnesium and protons: Design, synthesis, and properties of prototype struc-
tures. Biochemistry 1985, 19, 2396–2404.
16. London, R. E.; Levy, L. A.; Murphy, E. Fluorescent intracellular calcium
indicators. US Patent 5,516,911, 1996.
17. Tsien, R. Y.; Minta, A. Fluorescent indicator dyes for calcium working at
long wavelengths. US Patent 5,049,673, 1991.