Kinetic characterization of spiropyrans in aqueous media

Article abstract of DOI:10.1039/b818050d

Detailed kinetic investigations of the most common photoswitchable spiropyran, 6-nitro-BIPS, reveal that hydrolytic decomposition of its merocyanine isomer limits its utility in aqueous buffer; however, simple replacement of the 6-nitro substituent with a

Full text of DOI:10.1039/b818050d

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Kinetic characterization of spiropyrans in aqueous mediaw
Thorsten Stafforst* and Donald Hilvert
Received (in Cambridge, UK) 14th October 2008, Accepted 18th November 2008
First published as an Advance Article on the web 8th December 2008
DOI: 10.1039/b818050d
Detailed kinetic investigations of the most common photo-
switchable spiropyran, 6-nitro-BIPS, reveal that hydrolytic
decomposition of its merocyanine isomer limits its utility in
aqueous buffer; however, simple replacement of the 6-nitro
substituent with an 8-carboxylate yields a BIPS photoswitch
that is potentially better suited for biological applications.
Spiropyrans are valuable photochromic compounds that are
easily interconverted between colorless ring-closed spiropyran
(sp) and colored ring-opened merocyanine (mc) isomers either
by irradiation or heat (Fig. 1).1 They have been intensively
studied because of their utility in materials science.1 Although
biological applications are also desirable,2 relatively little is
known about the stability or kinetic properties of these dyes in
water.3 We have therefore examined the frequently used
6-nitro derivative of spiro-(2H-1-benzopyran-2,20-indoline)
Fig. 1 Isomeric forms of the spiropyran moiety embedded in
(BIPS) in aqueous solution to determine the relative rates of
thermal switching and decomposition.
peptides 1a and 1b. In aqueous solution the merocyanine form of
the chromophore undergoes hydrolysis to give Fischer’s base 2 and
salicylaldehyde 3.
Peptide 1a, which contains the 6-nitro-BIPS chromophore
and three lysine residues for solubility, was assembled by
Fmoc solid phase peptide synthesis.4 After HPLC purification,
it was isolated in the closed spiropyran form, 1asp, which has
an absorption maximum at 352 nm (Fig. 2). The open isomer,
1amc, was obtained by heating a millimolar solution of 1asp in
0.2% TFA to 90 1C for one minute and then diluting the
sample 100-fold in buffer. The merocyanine form of the dye
has absorbance maxima at 373 and 520 nm (Fig. 2). As
previously reported,1 the dye isomers are readily inter-
converted by light of different wavelengths. If an aqueous
solution of 1asp is irradiated at 350 nm, as is typically done,1
conversion to 1amc is only about 10%. However, irradiation
with UV light at 275 nm affords a 1 : 1 mixture of 1asp and
1amc within minutes, and irradiation of this mixture with
visible light (l = 520 nm) shifts the equilibrium back to the
closed form 1asp. A ca. 1 : 1 mixture of 1asp and 1amc is also
established thermally in buffer over several hours. Under these
conditions, however, decomposition of the merocyanine
isomer to give Fischer’s base 2 and 4-nitro-salicylaldehyde
3a competes with formation of the closed spiropyran (Fig. 1),
as established by LC-MS and absorption spectroscopy.
Hydrolysis is apparently initiated by conjugate addition
of water to the ene-iminium cation of 1amc, followed by a
retro-aldol reaction.
Fig. 2 Spectroscopic characterization of 1a. Absorption spectra: 1asp
(TT) and 1amc
(
); equilibrium mixture achieved by irradiation of 1a
at 275 nm ( ); thermal equilibrium established upon heating 1a for
5 min at 60 1C ( ); hydrolysis products obtained by heating the
sample at 60 1C for 90 min ( ). Fluorescence emission of 1amc
(520 nm excitation, ---). In each case, the concentration of peptide 1
was 50 mM in 10 mM sodium phosphate buffer, 100 mM NaCl, pH 7.0.
This system was characterized kinetically over a range of pH
values, starting from either 1asp or 1amc, by monitoring the
formation or disappearance of the merocyanine isomer at
520 nm (Fig. 3). The data were analyzed by fitting the
absorbance changes to the mechanistic model shown in
Fig. 1 using the program DynaFit.5 The rate constants for
thermal ring opening and ring closure are the same within
error over the pH range 5.0–8.0, and only about two times
faster than the pseudo-first order rate constant for hydrolysis
of 1amc (Table 1). The fact that hydrolysis of the merocyanine
(t1/2 E 4 h) occurs on roughly the same time scale as thermal
Laboratory of Organic Chemistry, ETH Zurich, Honggerberg HCI
¨
F322, CH-8093 Zurich, Switzerland.
¨
E-mail: stafforst@org.chem.ethz.ch; Fax: +41-44-632-1486;
Tel: +41-44-633-4198
w Electronic supplementary information (ESI) available: Experimental
section and data fitting. See DOI: 10.1039/b818050d
ꢀc
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Fig. 3 Typical time course of the thermal equilibration/degradation
of 1asp ) and 1amc ). Reactions were carried out with 20 mM
peptide in 10 mM sodium phosphate buffer, 100 mM NaCl, pH 7.0,
(
(
Fig. 4 Absorption spectra of 1bsp (TT) and 1bmc
(
) and fluorescence
emission and excitation spectra of 1bmc (440 nm excitation, ꢄ ꢄ ꢄ; 600 nm
emission, ---). The spectra were measured in 10 mM sodium phosphate
buffer, 100 mM NaCl, pH 7.0 with 25 mM peptide.
25.0 1C and monitored spectroscopically at 520 nm. The kinetic
parameters k1, k and k2 (Table 1) were obtained by fitting the data
ꢁ1
(---) to the model shown in Fig. 1.
but thermal reconversion to 1bmc occurs within minutes.
Table 1 Kinetic parameters for interconversion and degradationa
Although 1bmc decomposes at roughly the same rate as 1amc
,
the time scale of hydrolysis (t1/2 E 4 h) is much longer than
thermal ring opening (t1/2 E 1 min) or photochemical ring
closure (t1/2 E 1 min), allowing peptide 1b to be switched
many times between pure 1bsp and pure 1bmc with only minor
degradation. The large Stokes shift (160 nm) and the absence
of distinct features above 280 nm in the absorption spectra of
1bsp (Fig. 4) render this new photoswitch suitable for applica-
Peptide pH
k1/sꢁ1
k
ꢁ1/sꢁ1
k2/sꢁ1
K = k1/k
ꢁ1
1a
0.1% 8.0 ꢂ 10ꢁ5 o10ꢁ7
6.0 ꢂ 10ꢁ7 c1
TFA
5.0
6.0
7.0
8.0
7.0 ꢂ 10ꢁ5 7.0 ꢂ 10ꢁ5 2.0 ꢂ 10ꢁ5
1
1.1 ꢂ 10ꢁ4 1.1 ꢂ 10ꢁ4 2.0 ꢂ 10ꢁ5
1
8.0 ꢂ 10ꢁ5 9.0 ꢂ 10ꢁ5 4.0 ꢂ 10ꢁ5 0.9
1.0 ꢂ 10ꢁ4 2.6 ꢂ 10ꢁ4 6.0 ꢂ 10ꢁ5 0.4
1b
0.1% 1.6 ꢂ 10ꢁ2 n.d.
o10ꢁ7
tions involving Forster resonance energy transfer.
¨
TFA
5.0
6.0
7.0
8.0
1.4 ꢂ 10ꢁ2 n.d.
1.0 ꢂ 10ꢁ2 n.d.
1.0 ꢂ 10ꢁ2 n.d.
1.4 ꢂ 10ꢁ2 n.d.
5.5 ꢂ 10ꢁ6
In conclusion, relatively rapid hydrolysis of the mero-
cyanine form of 6-nitro-BIPS competes with thermal inter-
conversion of the open and closed forms of the dye under
physiological conditions, significantly limiting the utility of
this photochromic compound in water. Replacement of the
6-nitro substituent with an 8-carboxy group enhances the
rate of ring opening by two orders of magnitude, making
8-carboxy-BIPS an attractive alternative to 6-nitro-BIPS as a
chemical switch in biological systems.
5.0 ꢂ 10ꢁ5 4100
1.2 ꢂ 10ꢁ4
1.8 ꢂ 10ꢁ4
a
Reactions with peptides 1a (20 mM) and 1b (25 mM) were carried out
in 0.1% TFA–water or in 10 mM sodium phosphate buffer, 100 mM
NaCl, pH 5.0–8.0. Kinetic data were obtained from absorbance time
traces at 520 nm and 25.0 1C for 1a and 440 nm and 29.0 1C for 1b.
Estimated errors for all kinetic values are ꢃ20%.
This work was generously supported by the Deutschen
Akademie der Naturforscher Leopoldina (Bundesministerium
fur Bildung und Forschung BMBF-LPD 9901/8-158) and the
equilibration of the open and closed forms of the dye (t1/2 E 2 h)
seriously limits the utility of 6-nitro-BIPS as a photoswitch in
aqueous solution. Although the spiropyran form of the dye is
stable, decomposition begins as soon as 1amc is produced, either
photochemically or thermally, thus reducing the number of times
that 1asp and 1amc can be productively interconverted.
¨
ETH Zurich.
¨
Notes and references
1 (a) A. Samat, D. De Keukeleire and R. J. Guglielmetti, Bull. Soc.
Chim. Belg., 1991, 100, 679; (b) B. L. Feringa, Molecular Switches,
Wiley-VCH, Weinheim, 2001; (c) R. C. Bertelson, Organic
Photochromic and Thermochromic Compounds, ed. J. C. Crano
and R. J. Guglielmetti, Plenum Press, New York, 1999, vol. 1, p. 11.
2 (a) M. Inouye, Organic Photochromic and Thermochromic
Compounds, ed. J. C. Crano and R. J. Guglielmetti, Plenum Press,
New York, 1999, vol. 2, p. 393; (b) G. Mayer and A. Heckel, Angew.
Chem., Int. Ed., 2006, 45, 4900; (c) I. Willner and S. Rubin, Angew.
Chem., Int. Ed. Engl., 1996, 35, 367; (d) D. D. Young and A. Deiters,
ChemBioChem, 2008, 9, 1225; (e) L. Zhu, W. Wu, M.-Q. Zhu,
J. J. Han, J. K. Hurst and A. D. Q. Li, J. Am. Chem. Soc., 2007, 129,
3524.
To obtain dye molecules with more favorable properties for
biological applications, we prepared and isolated differently
substituted spiropyran derivatives by aldehyde exchange from
commercially available aldehydes. For example, peptide 1b,
containing an 8-carboxylic acid rather than a 6-nitro substi-
tuent, was generated by heating millimolar aqueous solutions
of 1a in the presence of a 10-fold excess of 3-formylsalicylic
acid. In contrast to 1a, which is isolated as the spiropyran 1asp,
peptide 1b is obtained as the merocyanine isomer 1bmc. In
aqueous buffer, the thermal equilibrium between 1bsp and 1bmc
lies completely on the side of the open merocyanine as a
consequence of a 125-fold faster rate of ring opening com-
pared to 1a (Table 1). The merocyanine can be quantitatively
converted to pure 1bsp by irradiation with blue light (440 nm),
3 (a) A. A. Garcia, S. Cherian, J. Park, D. Gust, F. Jahnke and
R. Rosario, J. Phys. Chem. A, 2000, 104, 6103; (b) K. Namba and
S. Suzuki, Bull. Chem. Soc. Jpn., 1975, 48, 1323.
4 T. Stafforst and U. Diederichsen, Eur. J. Org. Chem., 2007, 681.
5 P. Kuzmic, Anal. Biochem., 1996, 237, 260–273.
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This journal is The Royal Society of Chemistry 2009
288 | Chem. Commun., 2009, 287–288