Synthesis, photochromic properties, and light-controlled metal complexation of a naphthopyran derivative

Article abstract of DOI:10.1021/ol801406b

(Chemical Equation Presented) A light-controlled reversible binding switch based on photochromic 3H-naphtho[2,1-b]pyran is under development for studying cellular oscillatory calcium signals. The binding affinities of the closed and open forms of substituted naphthopyran 1 for Ca²⁺, Mg²⁺, and Sr²⁺ in buffer were determined. The photochemically ring-opened form of the receptor exhibited increased affinity compared to the thermally stable closed form of the receptor. The binding affinity difference for Ca ²⁺ was ～77-fold at pH 7.6.

Full text of DOI:10.1021/ol801406b

ORGANIC
LETTERS
2008
Vol. 10, No. 17
3761-3764
Synthesis, Photochromic Properties, and
Light-Controlled Metal Complexation of
a Naphthopyran Derivative
Satish Kumar, David Hernandez, Brenda Hoa, Yuna Lee, Julie S. Yang, and
Alison McCurdy*
CSULA Department of Chemistry and Biochemistry, 5151 State UniVersity DriVe,
Los Angeles, California 90032
amccurd@calstatela.edu
Received June 21, 2008
ABSTRACT
A light-controlled reversible binding switch based on photochromic 3H-naphtho[2,1-b]pyran is under development for studying cellular oscillatory
calcium signals. The binding affinities of the closed and open forms of substituted naphthopyran 1 for Ca2+, Mg2+, and Sr2+ in buffer were
determined. The photochemically ring-opened form of the receptor exhibited increased affinity compared to the thermally stable closed form
of the receptor. The binding affinity difference for Ca2+ was ∼77-fold at pH 7.6.
Calcium (Ca2+) is a second messenger in many cell types,
where it is used to translate extracellular signals into a wide
variety of intracellular events.1 Important cellular processes
are controlled by Ca2+, and many disease states are associated
with defects in the calcium signalosome. Manipulation of
Ca2+ concentration in cells through cage compounds is an
important tool for learning about this widespread signaling
system.2 Caged Ca2+ compounds undergo irreversible pho-
tochemical reactions that either release or take up Ca2+ when
triggered. However, cage compounds are less well suited to
the examination of oscillatory calcium signals, which may
encode information through both amplitude and frequency
modulation. The origin of these oscillatory signals is known,
but their effects at a molecular level are less well understood.
There is a need to develop new methodologies to study the
effects of spatiotemporal changes in Ca2+ concentration.
One approach to studying calcium oscillations is the
development of a water-soluble reversible small molecule
cage for Ca2+ triggered by light.3 To date, such a cage has
not been documented in the literature. In addition to
satisfying the design criteria for classic caged Ca2+,4
a
reversible binding photoswitch must exist in two intercon-
vertible forms with at least 10-fold difference in binding
affinity. The photoswitch must resist degradative processes
to mimic the wide variety of observed physiological repetitive
and oscillatory calcium signals. Photochromic compounds,
which can change structure dramatically and reversibly upon
(1) (a) Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Nat. ReV. Mol.
Cell Biol. 2003, 4, 517–529. (b) Carafoli, E. Trends. Biochem. Sci. 2004,
29, 371–379. (c) Rizzuto, R.; Pozzan, T. Physiol. ReV. 2006, 86, 369–408.
(d) De Koninck, P.; Schulman, H. Science 1998, 279, 227–230.
(2) (a) Furuta, T.; Noguchi, K. Trends Anal. Chem. 2004, 23, 511–519.
(b) Ellis-Davies, G. C. R. Methods Enzymol. 2003, 360, 226–238. (c)
Gurney, A. M.; Bates, S. E. Methods Neurosci 1995, 27, 123–152. (d) Davis,
J. P.; Tikunova, S. B.; Walsh, M. P.; Johnson, J. D. Biochemistry 1999, 38,
4235–4244. (e) Tompa, P.; To¨th-Bocona´di, R.; Friedrich, P. Cell Calcium
2001, 29, 161–170.
(3) (a) McCurdy, A.; Kawaoka, A. M.; Thai, H.; Yoon, S. C. Tetrahe-
dron Lett. 2001, 42, 7763–7766. (b) Lu, N. T.; Nguyen, V. N.; Kumar, S.;
McCurdy, A. J. Org. Chem. 2005, 70, 9067–9070.
(4) Lester, H. A.; Nerbonne, J. M. Annu. ReV. Biophys. Bioeng. 1982,
11, 151–175.
10.1021/ol801406b CCC: $40.75
Published on Web 07/23/2008
2008 American Chemical Society

irradiation, are well suited to this purpose. Photochromic
scaffolds are successfully incorporated into structures in
which chelation may be switched on and/or off through
irradiation.5 Naphthopyrans are a class of photochromic
compounds which show promise for this application.6
Irradiation of the colorless form with 360 nm light leads to
the cleavage of a C-O bond, resulting in colored isomeric
ring-opened forms which revert to the original form pre-
dominantly through thermal processes. Crown-ether-substi-
tuted naphthopyran scaffolds are effective binding photo-
switches for metal ions in organic solvents.7
Complexation of the closed form of 1 with metal ions was
examined spectroscopically. The UV-vis spectra of 1 show
minimal changes upon addition of excess Mg2+, Ca2+, and
Sr2+ (Figure S1, Supporting Information). The addition of
metal ions to the closed form of 1 did not result in any
detectable thermal ring opening induced by metal complex-
ation, unlike that observed for some photochromic
chelators.5d,7d,8 Small but significant and reproducible H
1
NMR shifts were observed upon addition of metal ions
(Table S2, Supporting Information). One or more aromatic
protons moved downfield upon addition of all metals to the
closed form of 1. The methylene protons only shifted when
calcium was added, and they moved upfield. The same
pattern of complexation-induced shifting, but with smaller
magnitudes, was also observed upon the addition of the same
three metals to phenyliminodiacetic acid in buffer (Table S3,
Supporting Information). The binding titration data were fit
to the two simultaneous binding equilibria shown below. An
additional equilibrium (1 chelator:2 metal ions) was consid-
ered but did not improve the fit to the data. The best fit
binding constants at two different pH’s are tabulated in Table
1 below. The pH of 7.6 was chosen to simulate physiological
environments as well as to completely deprotonate 1, closed.
A pH of 8.7 was also used in case the open form of 1 had
higher pKa’s. Clearly, each metal ion binds very weakly as
a 1:1 complex to the closed form of 1. The effect of pH on
the binding affinities is negligible. For Sr2+ at pH 7.6, the
binding affinities and the theoretical maximum upfield shifts
of 1 predicted by the binding model are inaccurate. This
inaccuracy is due to the difficulty determining a very small
binding affinity and the very small observable complexation
induced shifting. The relatively large binding constant K21
for all metals with 1 suggests that a second chelator binds
strongly with the 1:1 complex. This observation is consistent
with an incomplete coordination of the cations by the
iminodiacetic acid group and/or additional hydrophobic
association of the planar portion of the tricyclic ring systems
in the aqueous buffered solution.
Complexation of the open form of 1 with metal ions was
also examined spectroscopically. When 1 was irradiated with
UV light for 2 min in buffer, a visible absorption band
appeared (434 nm), which faded after irradiation was halted
(Figure 2). This long wave absorption corresponds to the
more conjugated open form of 1. The UV-vis spectra of
irradiated 1 in the presence of excess alkaline earth metal
ions were also recorded (Figure S2, Supporting Information).
A red shift (20 nm) of the long wave absorption band to
454 nm when irradiation of 1 occurred in the presence of an
excess of Ca2+ and a smaller red shift (7 nm) to 441 nm
occurred in the presence of an excess of Sr2+. The red shifting
of the long wave absorption of the open form of a
naphthopyran derivative upon addition of metal ions in
acetonitrile is also documented by other groups.7a,c The
shifting is thought to be indicative of stabilizing interactions
In the present work, we report the synthesis and charac-
terization of compound 1, a new water-soluble 3H-naph-
tho[2,1-b]pyran with an iminodiacetic acid substituent at
position 5 (Figure 1). This compound is designed so that
Figure 1. Two interconvertible forms of photochromic molecule
1. Open forms may adopt a cisoid or transoid configuration at the
indicated bonds.
the open form of 1 will exhibit a higher affinity for Ca2+
than the closed form. The binding affinities of closed and
open forms of compound 1 were determined, and the effects
of buffer composition and pH, on this reversible calcium
binding photoswitch were also examined.
Naphthopyran chelator 1 was obtained in five steps
(Scheme 1 and Supporting Information) with an overall yield
of 11%. Compound 1 is soluble up to ∼5.7 × 10-4 M in
aqueous solutions buffered at pH 8.7.
(5) (a) Kimura, K.; Sakamoto, H.; Nakamura, M. Bull. Chem. Soc. Jpn.
2003, 76, 225–245. (b) Sakata, T.; Jackson, D. K.; Mao, S.; Marriott, G. J.
Org. Chem. 2008, 73, 227–233. (c) Bren, V. A.; Dubonosov, A. D.; Minkin,
V. I.; Tsukanov, A. V.; Gribanova, T. N.; Shepelenko, E. N.; Revinsky,
Y. V.; Rybalkin, V. P. J. Phys. Org. Chem. 2007, 20, 917–928. (d)
Roxburgh, C. J.; Sammes, P. G. Eur. J. Org. Chem. 2006, 1050–1056. (e)
Strokach, Y. P.; Valova, T. M.; Barachevskii, V. A.; Shienok, A. I.;
Marevtsev, V. S. Russ. Chem. Bull., Int. Ed. 2005, 54, 1477–1480. (f) Lewis,
J. D.; Perutz, R. N.; Moore, J. N. J. Phys. Chem. A 2004, 108, 9037–9047.
(6) (a) Van Gemert, B. Benzo and naphthopyrans (Chromenes). In
Organic Photochromic and Thermochromic Compounds; Crano, J. C.,
Guglielmetti, R. J., Eds.; Plenum Publishing Corp: New York, N. Y., 1999;
Vol. 1, pp 111-140. (b) Moine, B.; Buntinx, G.; Poizat, O.; Rehault, J.;
Moustrou, C.; Samat, A. J. Phys. Org. Chem. 2007, 20, 936–943. (c)
Hepworth, J. D.; Heron, B. M. Prog. Het. Chem. 2005, 17, 33–62. (d)
Delbaere, S.; Micheau, J.-C.; Vermeersch, G. J. Org. Chem. 2003, 68, 8968–
8973.
(7) (a) Fedorova, O. A.; Maurel, F.; Chebun’kova, A. V.; Strokach, Y. P.;
Valova, T. M.; Kuzmina, L. G.; Howard, J. A. K.; Wenzel, M.; Gloe, K.;
Lokshin, V.; Samat, A. J. Phys. Org. Chem. 2007, 20, 469–483. (b) Ko˜szegi,
E.; Gru¨n, A.; Bitter, I. Supramol. Chem. 2006, 18, 67–76. (c) Ahmed, S. A.;
Tanaka, M.; Ando, H.; Iwamoto, H.; Kimura, K. Eur. J. Org. Chem. 2003,
2347–2442. (d) Stauffer, M. T.; Knowles, D. B.; Brennan, C.; Funderburk,
L.; Lin, F.-T.; Weber, S. G. Chem. Commun. 1997, 287–288.
(8) Chernyshev, A. V.; Metelitsa, A. V.; Gaeva, E. B.; Voloshin, N. A.;
Borodkin, G. S.; Minkin, V. I. J. Phys. Org. Chem. 2007, 20, 908–916
.
(9) Fedorova, O. A.; Strokach, Y. P.; Chebunkova, A. V.; Valova, T. M.;
Gromov, S. P.; Alfimov, M. V.; Loksin, V.; Samat, A. Russ. Chem. Bull.
Int. Ed. 2006, 55, 287–294
.
3762
Org. Lett., Vol. 10, No. 17, 2008

Scheme 1. Synthesis of 1
between metal cation and oxygen that are enhanced in the
presumably more polar excited state. Here, the absence of a
red shift of the open form of 1 in the presence of Mg2+
suggests there is little interaction between this metal and the
oxygen. The different absorbance values at the λmax, despite
constant chelator concentration, may be attributed to one or
more of the following: a change in thermal fade rate constant
in the presence of metal ions (see below), a difference in
the extinction coefficients of metal-bound 1, a change in the
distribution of geometric isomers of 1 in the presence of
different metals, and variable irradiation intensities.
Rates of thermal closure (k∆) of open forms of 1 to the
closed form were determined in the presence of metal ions
(Tables S4-S6, Supporting Information). Ca2+ addition
decreased the observed rates of thermal closure of 1 at two
different pH values. Sr2+ had this effect at pH 8.7 as well,
although less pronounced than for Ca2+. By contrast, the
addition of Mg2+ increased the rate of thermal closure at
pH 8.7 and had little effect on rates at pH 7.6. The increase
of thermal fading rates of the open form of a crown-
substituted naphthopyran by Mg2+ in acetonitrile has been
documented previously,9 but in that case, a red shift upon
addition of Mg2+ was observed, in contrast to no observable
shift of 1 here. To ascertain the effect of ionic strength
changes on thermal fading kinetics, KCl was added to
irradiated solutions of 1 and did not result in any changes in
fading rates (Table S7, Supporting Information). Therefore,
specific interactions between the divalent metal ions and the
open forms of 1 must account for the observed rate changes.
The observed changes in rates of thermal closure may be
interpreted most simply as a result of stabilization (slowing
thermal closure) or destabilization (increasing thermal closure
rates) of the open forms by metals. Alternatively, the addition
of metals may affect the distribution of geometric isomers
formed upon irradiation, each isomer having a different
fading rate.
The open form of 1 is short-lived, which prevents the use
of a binding titration to determine metal binding affinities.
However, the kinetics of thermal closure in the absence and
presence of metal ions may be monitored to determine the
binding equilibrium constant of the open form of a photo-
Table 1. Best Fit Binding Constants (K11, K21) and Maximum
Shift (∆δmax) for Chelator 1 in 10 mM Tris by H NMR
a
1
K11 (M-1
)
K21 (M-1
pH 8.7
)
∆δmax (Hz)d
Mg2+ 1.20 ( 0.12 × 101
Ca2+ 4.07 ( 0.73 × 101
2.24 ( 0.44 × 105
9.3 ( 1.8b
18.0 ( 2.2b
8.5 ( 1.0b
7.77 ( 1.05 × 104
Sr2+
3.16 ( 0.51 × 10 ° 3.23 ( 0.62 × 104
pH 7.6
Mg2+ 7.57 ( 0.60 × 10 ° 3.45 ( 0.34 × 105
7.9 ( 0.6c
Ca2+ 3.10 ( 0.35 × 101
5.10 ( 0.20 × 104
13.8 ( 0.2c
Sr2+
6.55 ( 0.54 × 10-2 6.77 ( 1.31 × 104 389.5 ( 188.9c
a Average of 3 trials. [1]0: 1.97 × 10-4 M; [M2+] ) 0∼0.17 M.
b Both 400 and 600 MHz instruments were used. c Obtained on a 600
MHz instrument. d ∆δmax values are reported for the aromatic proton “b”
(Table S2). The ∆δmax obtained by the 600 MHz instrument were divided
by 1.5.
Figure 2
.
Thermal fading of 1 in the dark over time. [1] ) 1.70 ×
10-5 M in 10 mM Tris, pH 8.7, at 24 °C.
Org. Lett., Vol. 10, No. 17, 2008
3763

chromic compound.10 Using this procedure, a graphical
method was used to obtain the binding affinity of the open
form of 1 with metal cations (Figures S4-S9, Supporting
Information). The resulting binding affinities between Ca2+
and Sr2+and the open form of 1 at different pH are shown
in Table 2. The effect of pH on the affinities was negligible.
Table 2. Metal Binding Affinities of the Open Form of 1 in 10
mM Tris at 24 °C
Keq (M-1
)
Figure 3. Five cycles of 2 min of UV irradiation of 1 ([1] ) 1.70
metal ion
Ca2+
pH
× 10-5 M) followed by thermal (dark) closure in 10 mM Tris, pH
8.7, and 24 °C.
8.7
7.6
8.7
2.53 ( 0.24 × 103
2.39 ( 0.26 × 103
5.94 ( 0.48 × 102
Sr2+
In summary, a water-soluble photoswitch for Ca2+ has
been synthesized and characterized. Compound 1 exhibited
a 77-fold affinity difference between closed and open forms,
which is promising for a metal binding photoswitch. To the
best of our knowledge, this is the first demonstration of a
water-soluble, small molecule reversible photoswtich for
Ca2+ binding. To achieve the binding affinity and oscillation
periods more appropriate for intracellular conditions, further
tuning of binding site geometry and switching kinetics
through structural modifications of 1 is in progress. The work
reported here represents a significant step in the development
of a practical reversible cage for Ca2+ that will find use in
investigations of intracellular calcium signaling.
The faster rates observed in the presence of Mg2+ are
incompatible with using this kinetic model for binding
constant determination. As anticipated, the binding affinities
for both Ca2+ and Sr2+ were significantly different for the
closed versus open forms of 1. For Ca2+, there was a 77-
fold enhancement in affinity upon irradiation at pH 7.6 and
a 62-fold enhancement at pH 8.7.
The effects of pH and buffer salt composition on photo-
chromism were determined for five cycles of irradiation and
thermal closure. An example of photoswitching in 10 mM
Tris, pH 8.7, is shown in Figure 3 below. Changing pH
(7.6-9.8) and buffer salt identity (phosphate, HEPES) did
not significantly affect photoswitching (Figures S13-S16,
Supporting Information). Addition of 4 mM Ca2+ to the
solution had little effect on the absorbance, except for a slight
diminishment of the absorbance at the photostationary state
(Figure S17, Supporting Information). Addition of Mg2+
resulted in a significant decrease of the absorbance at the
photostationary state due to the increased rate of thermal
closure (Figure S18 and S19, Supporting Information).
Acknowledgment. This work was supported by a grant
from the National Institutes of Health (NIGMS MBRS S06
GM08101). The authors are thankful to P. Britell, N.
Heckmann, and K. Miller (CSULA Department of Chemistry
and Biochemistry) for their assistance.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(10) (a) Zhou, J.; Zhao, F.; Li, Y.; Zhang, F.; Song, X. J. Photochem.
Photobiol. A 1995, 92, 193–199. (b) Flannery, J. B. J. Am. Chem. Soc.
1968, 90, 5660–5671.
OL801406B
3764
Org. Lett., Vol. 10, No. 17, 2008