1. Multiresponsive Switchable Diarylethene
ORGANIC
LETTERS
2009
Vol. 11, No. 17
Multiresponsive Switchable Diarylethene
and Its Application in Bioimaging
Xijun Piao, Ying Zou, Junchen Wu, Chunyan Li, and Tao Yi*
Department of Chemistry and Laboratory of Advanced Materials, Fudan University,
Shanghai , P.R. China

2. Introduction
Fluorescence labeling technology is important to the advancement of biological imaging, especially for the monitoring of internal cellular processes.
Lots of work has been carried out, in designing fluorescent probes for bioimaging and some materials, like organic dyes, fluorescent proteins…etc are now widely using standards.
But in case of cellular imaging most of these probes respond
irreversibly to a certain event or nondynamically to environmental stimuli.

3. Chem. Commun., 2007, 4758–4760
A switchable fluorescent organogel (orange fluorescence, both in solution and in gel state) containing a photochromic diarylethene unit, and a bisamido cyclohexane unit (racemic) for the capacity to gelate organic solvents by intermolecular hydrogen bonding.
This fluorescence could be switched by alternate UV and visible light irradiation both in solution and in the gel state.
Microsized erasable fluorescent images of compound on a gel (20mgml-1
in acetonitrile) using confocal laser scanning microscopy. (Written (W)
and rewritten (RW) by a 405 nm laser for 10 min, erased (E) with 550 nm
light for 1 h; fluorescence readout by a 488 nm laser. Scale bar: 20 mm).

4. In their study they found that, photochromic organogel based on diarylethene, whose optimal excitation wavelength (470 nm) would not destroy the structures of both open and closed isomers of diarylethene. Therefore, this system is suitable for application in a non-destructive fluorescent readout switch for storage.
Compounds such as rhodamine and cyanine dyes, are useful for cellular studies but can respond only irreversibly to one event.
Though several photoactivatable fluorescent proteins have been developed, but due to the large size of fluorescent proteins often results in improper protein functioning.
Li et al.* reported that a dual-color fluorescent probe based on spiropyran loaded nanoparticles, but the irradiation source provides shorter than 365 nm UV light and as we knows the thermal instability of the open-ring form of spiropyran limited its application in bioimages.
It is well-known that diarylethene is the most promising switchable unit within those classical photochromic systems owing to its excellent fatigue resistance and thermal stability. However, most of these studies are carried out in organic systems, including the fluorescent photochromic diarylethene for labeling biomolecules by M. Irie**
*Li, A. D. Q. J. Am. Chem. Soc. 2007, 129, 3524–3526.
**Soh, N.; Yoshida, K.; Nakajima, H.; Nakano, K.; Imato, T.; Fukaminato, T.; Irie, M. Chem. Commun. 2007, 5206–5208.

5. These problems may be resolved by inducing amphiphilic molecules, which is a widely used technique to construct highly organized nanostructures in aqueous systems with the driving force of hydrophobic interaction.
This group construct an amphiphilic molecule with hydrophilic and hydrophobic chains at two ends of a rigid diarylethene core. It forms stable vesicles in water and exhibits different fluorescence in open and closed states under alternating irradiation of UV and visible light. Thus, this molecule would serve well as a photoswitchable probe for imaging living Cells.
CLSM image (above) and the overlay image (bottom) of KB* living cells incubated with compound for 20 min at 25 °C (1) in original state, (2) irradiated by 405 nm light (2 mW) for a single cell, (3) all cells, and (4) recovered by 633 nm light (0.7 mW) (λex ) 405 nm, 0.15 mW).
*KB cells (A cell line derived from a human carcinoma of the nasopharynx, used as an assay for antineoplastic agents) using confocal laser scanning microscopy (CLSM).

6. Present work…
Limitation of their previous work was , it could only use for static
imaging and couldn’t use as a probe that showing biological process.
Combination of diarylethene and terpyridine functional units .
Exhibits several clearly different and reversible fluorescence states which can be controlled by varying light frequency and metal ion concentration.
Functioning well in solution, so can apply in biological systems too.
When stimulated by metal ions its showing easily detectable changes in fluorescence and thus can serve well as a photoswitchable probe for imaging live cells and previously nonvisualizable biological processes.

7. Synthesis…

8. Absorption and fluorescence emission changes of compound in THF (110-5 M) with 365 nm light .
A single absorption band in the ultraviolet range (300nm) corresponds to π-π* transition.
Blue fluorescence centered at 440 nm with
fluorescent quantum yield was when using Rhodamine B as reference.
When irradiated with UV(365 nm), the colorless
solution turned slightly purple within 4 min and a new absorption maximum appeared at 550 nm.
When irradiated with Visible(549 nm) fluorescence intensity was almost restored to the original state.
The difference in absorbance at 550 nm for the open isomer and the PSS was less than 2% and 8%, respectively, after the open-close cycle was repeated 5 times by alternating irradiation with UV and visible light .This indicated that compound possessed good fatigue-resistance. So we can use this compound in a repeated “write-erase” Process.

9. Coordination with metals…
It can coordinate with both Zn2+ and Cu2+, which will decrease the fluorescent intensity.
Alkali and alkaline earth metal cations such as Na+, Mg2+gave no interference at a 3.5-fold excess concentration.
Transition metal and heavy-metal ions such as Fe2+, Hg2+ gave a weak response .
Zn2+ was chosen into the quenching of the fluorescence because
aqueous solutions of Zn2+ are usually colorless and it is the strongest Lewis acid among divalent metal ions.
By the addition of aqueous solution of Zn(NO3)2, fluorescent intensity was quenched which is due to the formation of a compound-Zn complex, changing the charge density of the terpyridine unit.
When EDTA solution (1.0 eq to Zn2+)was added, the fluorescence intensity was restored which is due to the difference in the association constant between EDTA-Zn
and compound -Zn. [The KEDTA-Zn (3.16 × 1016 M-1) is >>Kcompound-Zn (1.76 × 106 M-1)].
Therefore, Zn2+ acts as a trigger for fluorescence switch-OFF, while EDTA acts as a trigger
for fluorescence switch-ON.
From this we can conclude that the fluorescence intensity of compound can be reversibly controlled by UV/visible light or Zn/EDTA, and this compound can acts as a double-controlled molecular fluorescence switch reacting to light and chemical stimuli.

10. Fluorescence intensity changes at 440 nm with different molar ratio of Zn2+ and compound in THF/ water solution (100:1, v/v) (compound from 2×10-5 to 1×10-6 M, Zn2+ with the fixed concentration of 1×10-5 M). (Inset) Fluorescence spectral changes of compound (1×10-5 M) by the addition of Zn2+ from 0 to 3.5 equiv.

11. When incubated with PBS/DMSO (100:2, v/v) solution of the compound(1×10-5 M) for 20 min at 25C observed a blue luminescence.
The luminescence could be controlled by using UV/ visible light as the switching trigger.
selected cell
405 nm for 3 min
633 nm for 40 min
Overlay image
A& F- Bright field transmission image.
B- Overlay image of A and C.
Confocal fluorescence image of (C) original state, (D) irradiated
by 405 nm light (2 mW, 3 min) for one selected cell and (E)
recovered by 633 nm light (0.7 mW, 40 min). Confocal fluorescence
image of (G) original state of F, and incubation by Zn2+ solution
with the concentrations of (H) 5 × 10-5 M, (I) 1 × 10-4 M. (J)
Recovered by 5 × 10-4 M EDTA solution. (K), (L), (M), (N) were
the distribution of fluorescence indensity of (G), (H), (I), (J),
With different Zn2+ conc:
After addition of EDTA solution
510-5M Zn2+
110-4M Zn2+
510-4M EDTA

12. Cytotoxicity Assay.
By means of MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} assay.
Concentration of compound/ μM
The cellular viabilities were found to be greater than 85% after 24 h in the presence of compound at various concentration.
This indicates that compound has low cytotoxicity.

13. Conclusion…
They have developed a new multiresponsive fluorescence switch based on diarylethene and terpyridine units.
It exhibits effective switchable fluorescence controlled by UV/visible light or metal ions/EDTA in solution.
It have low cytotoxicity, so can enter live cells as a fluorescence probe and can act as a detector for the biological process of the metal ion transmembrane transport.

14. thank you…