A new class of azobenzene chelators for Mg2+ and Ca 2+ in buffer at physiological pH

Article abstract of DOI:10.1016/S0040-4039(03)01863-X

A new class of azobenzene-based chelators, trans-3a and trans-3b (3a and 3b), were designed and synthesized in two steps. Both 3a and 3b were readily dissolved in a buffer solution at physiological pH. The values of the dissociation constant of 3a and 3b for Mg²⁺ and Ca²⁺ were determined by the Hills plot; K_d^Mg=1.12 mM and K _d^Ca=660 μM for 3a and K_d^Mg=158 μM and K_d^Ca=200 μM for 3b, respectively. On irradiation at 489 nm light, 3a isomerized to give cis-form, which underwent cis-to-trans thermal isomerization in darkness at room temperature. The change in the absorption spectrum of the irradiated solution of 3a in the presence of Mg²⁺, showing the cis-to-trans thermal isomerization, indicates that the affinity of cis-3a for Mg²⁺ is lower than that of 3a.

Full text of DOI:10.1016/S0040-4039(03)01863-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 7277–7280
A new class of azobenzene chelators for Mg2+ and Ca2+ in
buffer at physiological pH
Atsuya Momotake and Tatsuo Arai*
University of Tsukuba, Tennnodai 1-1-1, Tsukuba 305-8571, Japan
Received 11 July 2003; revised 25 July 2003; accepted 1 August 2003
Abstract—A new class of azobenzene-based chelators, trans-3a and trans-3b (3a and 3b), were designed and synthesized in two
steps. Both 3a and 3b were readily dissolved in a buffer solution at physiological pH. The values of the dissociation constant of
3a and 3b for Mg2+ and Ca2+ were determined by the Hills plot; KdMg=1.12 mM and KdCa=660 mM for 3a and KdMg=158 mM
and KdCa=200 mM for 3b, respectively. On irradiation at 489 nm light, 3a isomerized to give cis-form, which underwent
cis-to-trans thermal isomerization in darkness at room temperature. The change in the absorption spectrum of the irradiated
solution of 3a in the presence of Mg2+, showing the cis-to-trans thermal isomerization, indicates that the affinity of cis-3a for Mg2+
is lower than that of 3a.
© 2003 Elsevier Ltd. All rights reserved.
Azobenzene and its analogues play an important role
for a tremendous amount of functionalized molecules
due to its efficient photo trans-cis and thermal cis-to-
trans isomerization properties. In addition, many of the
spectrophotometric reagents for detection of a metal
ion in aqueous solution contain azobenzene skele-
tons.1–3 Azobenzenes are key molecules for creating
new functionalized molecules because (1) they are
potential good chromophore and (2) they possess
photo- and thermal-switchable properties. In this con-
text basic studies of their photochemical behavior and
of the structure–spectral relationship are essential for
development of a new class of functionalized azoben-
zene derivatives. As a part of our research, we previ-
ously reported azobenzene dendrimers where the
azobenzene works as a dendrimer core, to investigate
the effect of a bulky dendron subunit on the isomeriza-
tion properties.4 In this study we focus our attention on
the azobenzene as a potential good chromophore to
develop photofunctionalized molecules such as caged
calcium controlled by light.5–7 We describe here the
design, synthesis, photochemistry and binding proper-
ties of biologically important metals of a new class of
water-soluble azobenzene chelators. The effect of
methyl groups in the azobenzene ring on the binding
and photochemical behavior is also incorporated.
For designing the azobenzene chelators, the following
points were considered: (1) Absorption properties: If a
chelator absorbs visible light, it could be a potential
color indicator. In order to obtain an azobenzene
derivative with large extinction coefficient at longer
wavelength, electronic push–pull effect on the aromatic
Scheme 1. Reagents and conditions: (a) Bromoethyl acetate,
NaI, (i-Pr)2NEt, CH3CN, reflux, 3–5 days; (b) KOH, EtOH,
12 h; (c) MgCl2, HEPES, KCl buffer, pH 7.2, 22°C.
* Corresponding author. Tel.: +81-298-53-4315; fax: +81-298-53-6503;
e-mail: arai@chem.tsukuba.ac.jp
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01863-X

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A. Momotake, T. Arai / Tetrahedron Letters 44 (2003) 7277–7280
ring will be helpful so that an electron-donating group
should be introduced in the ortho- or para-position to
the azo linkage; (2) Solubility: Water-solubility at phys-
iological pH is necessary for chelating reagents if the
targets are physiologically important metals, such as
calcium and magnesium. In some cases, the binding
constants for Ca2+ or Mg2+ are determined in an
organic solvent8 or a mixture of water–organic solvent
probably because of the poor water-solubility of chela-
tors.9 Generally, the binding affinity is strongly affected
by the solvent polarity, temperature, pH, and so forth.
Therefore, before preparation, it should be considered
that the chelators which are not water-soluble are
hardly any help to measure the amount of the metals in
aqueous solution; (3) Binding properties: Actually, it is
difficult to estimate the binding affinity of the chelators
for each metal before they are prepared. As the first
series of compounds for this study, we chose the
EDTA-like structures trans-3a and trans-3b (3a and 3b)
because it would increase not only the binding affinity
for Ca2+ and Mg2+ but also water solubility as well as
the electron-donating effect on the aromatic ring.
The chelator 3a and its methyl derivative 3b were
obtained in two steps (Scheme 1), starting from 1a10
and 1b11 which were prepared by KO2 oxidation of
o-phenylenediamine for 1a and of 4,5-dimethyl-1,2-
phenylenediamine for 1b, respectively. Alkylation of 1a
and 1b with ethyl bromoacetate was achieved in reflux-
ing acetonitrile in the presence of N,N-diisopropylethy-
lamine and sodium iodide to give tetraalkylated
azobenzene 2a and 2b, respectively, in moderate yield.
Compounds 2a and 2b were saponified in aqueous
KOH–ethanol. After the hydrolysis completed, the sol-
vent was removed by evaporation and the crude
product was dissolved in hot ethanol and diethyl ether
was added. The mixture was allowed to stand for 30
min at room temperature to give a reddish-orange
precipitate, which was an almost pure product. The
obtained potassium salts 3a and 3b were characterized
by UV, NMR and elemental analysis. Both compounds
readily dissolved in water at physiological pH to give
the red solution. The all products obtained in this
procedure were trans-isomer, which were determined by
1H NMR. cis-Isomers were not obtained probably
because they were unstable to go back to corresponding
trans-isomers at room temperature. The structure of
Mg2+-bound form of 3a shown in Scheme 1 is tentative.
Figure 1. Change in absorption spectra of 3a (a) and 3b (b)
upon the addition of MgCl2 in 40 mM HEPES, 100 mM KCl
buffer, pH 7.2, 22°C: before titration (dash-dot line) and
excess MgCl2 (solid line). Inset shows the similar spectral
change upon the addition of CaCl2.
for 3a and 475 and 343 nm for 3b, respectively. A series
of spectra were conveniently analyzed by the Hill plot,
i.e. a plot of log[(A−A0)/(A1−A)]=y versus log[Mg2+]=
x. A0 is the absorbance of the free tetraanion before
titration, A1 is the absorbance of the magnesium com-
plex and A is the absorbance at intermediate magne-
sium level. The data were fit to a straight line and the
UV absorption spectra of 3a and 3b at various levels of
free Mg2+ were monitored at room temperature as
shown in Figure 1. In the absence of divalent ions, the
spectrum shows a maximum at 489 and 316 nm for 3a
and 505 and 334 nm for 3b, respectively in a buffer
solution (40 mM Hepes/100 mM KCl, pH 7.2 at 22°C).
The absorption maximum of 3b slightly red-shifted by
the effect of introduction of methyl groups in the
para-position of azo-linkage. To the solution of the free
chelator, increasing amounts of the MgCl2 solution
were added and after each titration step, absorption
spectra were recorded. Magnesium ion binding to the
chelator causes a major hypsochromic shift toward a
limiting spectrum with a maximum at 473 and 322 nm
Mg
value for Kd could be determined: 1.12 mM for 3a
Mg
and 158 mM for 3b, respectively. The Kd value for 3b
is approximately seven times lower than that of 3a
suggesting that the binding affinity for Mg2+ is affected
by the change in the electronic environment on the
azobenzene aromatic ring due to the two methyl
groups. The dissociation constant for Mg2+ found for
3a in vitro is similar to the values reported for APTRA
(aminophenol triacetic acid)-based magnesium chela-

A. Momotake, T. Arai / Tetrahedron Letters 44 (2003) 7277–7280
7279
tors.12,13 Since the values correspond well with the low
and submillimolar levels of cytosolic free Mg2+, the
chelators 3a and 3b may be used as the non-fluorescent
colorimetric reagents for Mg2+ under physiological con-
ditions. The dissociation constants for Ca2+ were also
determined by the same method and were 660 and 200
mM for 3a and 3b, respectively. Similar spectral changes
of 3a and 3b upon the addition of CaCl2 are shown in
the inset of Figure 1. It should be noted that 3a and 3b
have higher selectivity for Mg2+ than for Ca2+ in intra-
cellular fluid because the basal cellular free Ca2+ level,
about 100 nM, is much lower than that of Mg2+. This
indicates that Ca2+ does not interfere with Mg2+ mea-
surement under normal conditions in intracellular fluid.
are characteristic of trans–cis isomerization of an
azobenzene chromophore. The inset in Figure 3 shows
the time profile of photo trans–cis isomerization (ꢀ) on
irradiation at 489 nm light and thermal cis-to-trans
isomerization (ꢁ) of 3a at 23°C in darkness. Methyl
derivatives 3b, on the other hand, did not isomerize on
irradiation at 510 or 365 nm light in a buffer at pH 7.2.
The present finding indicates that the introduction of
electron-donating groups in the para-position of azo-
linkage, in addition to the ortho-amino groups, proba-
bly increases the intramolecular charge transfer and
affects the singlet excited state behavior, to result in
change of the photoisomerization properties. Ca2+- or
Mg2+-bound form of 3a after titration also did not
exhibit photoisomerization on irradiation at 489 or 365
nm light.
Since compounds 3a and 3b are azobenzene analogues,
photoisomerization was tested (Scheme 2). On irradia-
tion at 489 nm light, the absorbance at 489 nm of 3a
decreased with time. Figure 2 shows the change in the
absorption spectra of 3a. Similar spectral change was
observed on irradiation at 365 nm light. The ratio of 3a
and cis-3a at the photo-stationary state was ([trans]/
[cis])pss=80/20, determined by 1H NMR. When the
irradiated solution of 3a was kept in darkness at room
temperature, the absorbance at 489 nm increased and
was mostly back to that of 3a after 3 h with the rate
constant of 1.43×10−2 s−1 at 22°C. These spectral
changes with photoirradiation as well as dark reaction
The difference in affinities of 3a and cis-3a for Mg2+
was investigated. Figure 3 shows the change in the
absorption spectra of 3a. The absorbance of 3a, 5.32×
10−5 M in HEPES, KCl buffer, pH 7.2, 22°C (dotted
line) decreased with irradiation at 489 nm light to give
the trans–cis photostationary state (line not shown).
Then, the absorbance of the irradiated solution was
further decreased by addition of MgCl2 (3.32×10−5 M)
(thin line). Interestingly, the absorbance, observed just
after adding MgCl2, increased with time. When 160 min
passed after addition of MgCl2, the increasing spectra
(ꢀ) overlapped with another spectrum of 3a in the
Scheme 2. Photo trans–cis and thermal cis-to-trans isomeriza-
tion of 3a.
Figure 3. Absorption spectra of 3a before irradiation in
HEPES, KCl buffer, pH 7.2, 22°C (dotted line), after addition
of MgCl2 (3.32×10−5 M) without irradiation (solid line), right
after addition of MgCl2 (3.32×10−5 M) to the irradiated
solution (thin line), and 160 min later after addition of MgCl2
to the irradiated solution (ꢀ). Inset shows the time depen-
dence of the absorbance change for irradiated solution of 3a
with added MgCl2 (3.32×10−5 M), recorded at 489 nm for
thermal cis-to-trans isomerization in the dark at 22°C (ꢁ)
and absorbance of non-irradiated 3a solution with added
MgCl2 (3.32×10−5 M) (ꢀ).
Figure 2. Change in absorption spectrum of trans-3a on
irradiation at 489 nm in HEPES, KCl buffer, pH 7.2, 22°C.
Inset shows the time dependence of the absorption change at
489 nm for photo trans–cis isomerization (ꢀ) and thermal
cis-to-trans isomerization in the dark at 23°C (ꢁ).

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A. Momotake, T. Arai / Tetrahedron Letters 44 (2003) 7277–7280
presence of MgCl2 (3.32×10−5 M) (solid line). The result
indicates that the affinity of the cis-isomer for Mg2+ is
much lower than that of the trans-isomer (Scheme 3).
The inset in Figure 3 shows the increase of the
absorbance for the irradiated solution of 3a with MgCl2
(3.32×10−5 M), recorded at 489 nm, exhibiting the
thermal cis-to-trans isomerization (ꢁ) and the
absorbance of non-irradiated 3a with MgCl2 (3.32×10−5
M) (ꢀ).
1.13 (12H, t, J=7.1 Hz, CH3). ESI-MS: calcd for
C32H44N4NaO8: [M+Na]+; m/z 635.3051. Found:
1
635.2949. 3a: H NMR (400 MHz; D2O) l 7.56 (2H, d,
J=7.7 Hz, ArH), 7.30 (2H, t, J=7.7 Hz, ArH), 6.88–
6.82 (4H, m, ArH), 4.06 (8H, s, NCH2). Anal. calcd for
C20H16K4N4O8·9H2O·KOH: C, 29.47; H, 4.33; N, 6.87.
1
Found: C, 29.54; H, 4.11; N, 6.74. 3b: H NMR (400
MHz; D2O) l 7.41 (2H, s, ArH), 6.68 (2H, s, ArH),
4.40 (8H, s, NCH2), 2.25 (6H, s, CH3), 2.20 (6H, s,
CH3). Anal. calcd for C24H24K4N4O8·9H2O: C, 35.37;
H, 5.19; N, 6.87. Found: C, 35.59; H, 5.18; N, 6.82.
Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Areas (417) and the 21 Cen-
tury COE Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of
the Japanese Government and by the Asahi Glass
Foundation.
Scheme 3.
In summary, a new class of azobenzene-based chelators
3a and 3b was designed and synthesized by simple
reactions and their basic properties were described.
Both 3a and 3b were readily dissolved in water at
physiological pH. Introduction of methyl groups in the
aromatic ring affected the dissociation constant for
metals as well as the photoisomerization properties. On
irradiation at 489 nm light, 3a isomerized to give
cis-form, which underwent cis-to-trans thermal isomer-
ization in darkness at room temperature. The experi-
mental results on the absorption spectra of the
irradiated solution of 3a with Mg2+ indicate that the
affinity of cis-3a for Mg2+ is much lower than that of
3a. To the best of our knowledge, this is the first clear
example to prepare photoresponsive azobenzene chela-
tors where the affinity for Mg2+ is different between the
trans- and cis-isomers. In addition, 3a and 3b can be
used as a magnesium indicator under physiological
conditions.
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2a: H NMR (400 MHz; CDCl3; Me4Si) l 7.23 (2H,
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