Fluorescent Sensors for Zinc (II) Detectionin Biological Systems

Stephen F. LincolnDepartment of Chemistry, University of Adelaide, SA 5005

SHOWCASE ON RESEARCH

Vol 35 No 3 December 2004 AUSTRALIAN BIOCHEMIST Page 13

Two to three grams of zinc are contained in the human body as Zn2+ where it is the second most common transition metal after iron (Fe2+/Fe3+). Very little Zn2+ appears to exist in its fully hydrated form, [Zn(OH2)6]2+, and in mammalian and bacterial cells it is predominantly bound by peptides and proteins. In this state, it acts as a catalytic centre (e.g. carbonic anhydrase) , an enzyme regulator (e .g. alkaline phosphatase), a structural cofactor (e.g. Cu-Zn SOD), or functions in DNA binding and recognition processes (e.g. zinc-finger proteins), and in neurological signal transmission. Variations in Zn2+ levels in tissues are associated with wound healing and a range of diseases exempli f ied by ischemia, epi lepsy, s troke and Alzheimer's disease. As a consequence, the detection and localization of Zn2+ in tissues and in complete organisms is of great interest and has been the focus of c o n s i d e r a b l e e f f o r t . T h e f i e l d h a s b e e n comprehensively reviewed by Jiang and Guo (1).

Zn2+-Selective Fluorescent SensorsWhile atomic absorption spectroscopy and other

analytical methods provide very accurate quantitative determinations of [Zn2+], they necessarily destroy the mater ia l analysed. In contrast , a f luorophore-containing molecule, or sensor, which undergoes either a fluorescence intensity or wavelength change on binding Zn2+ provides both the location of Zn2+ and a semi-quantitative measure of [Zn2+] for in vivo and in vitro samples. However, the sensor must be highly Zn2+ selective. This requires that it binds Zn2+ much more strongly than Na+, K+, Mg2+ and Ca2+ which are all-pervasive in biology. This is achieved by designing the sensor to bind Zn2+ strongly through donor groups such as sulfonamides and amines which bind the alkali metal and alkaline earth metal ions much less effectively. Nevertheless, the first row metal ions Mn2+, Fe2+, Co2+, Ni2+ and Cu2+ are still strongly bound by sensors designed in this way. Fortunately, these metal ions have five to nine electrons in their d orbitals and transitions between their different energy states provide pathways for quenching fluorescence such that the bound sensor does not fluoresce. In contrast, Zn2+ has ten d electrons which greatly limits this mechanism for fluorescence quenching such that the bound sensor either fluoresces, or undergoes a fluorescence intensity or wavelength change if it is already fluorescent in the unbound state. This leaves Cd2+, a heavy metal ion in the same group as Zn2+ which also has ten d electrons. Cd2+ usually binds Zn2+-binding sensors less strongly than Zn2+, but it modifies their fluorescence in a similar manner.

However, Cd2+ is very toxic and is unlikely to exist at levels which interfere with Zn2+ detection in most biological samples.

T h e O p e r a t i o n o f a Z n 2 + - S e l e c t i v e Fluorescent SensorA widely used Australian developed Zn2+-selective

s e n s o r , Z i n q u i n E ( [ e t h y l ( 2 - m e t h y l - 8 - { p -tolylsulfonylamino}-6-quinolyloxy]acetate), is used to illustrate the basic principle of the operation of Zn2+ sensors in Fig. 1 (2). The design of a Zn2+ sensor for biological use requires it to be reasonably water soluble and to be sufficiently membrane soluble to enter biological cel ls . Thus, to provide water solubility it will generally possess one or more hydrophilic groups attached to hydrophobic aromatic groups which provide membrane solubility and also have significant conjugation in their π bonding to ensure fluorescence when bound to Zn2+. Zinquin E possesses these characteristics and its uncharged nature is considered to enhance its ability to traverse a cell membrane. Intracellularly, it is expected that esterases will hydrolyse the ester group to give the carboxylic acid, Zinquin A, which at physiological pH exists in the carboxylate form, the negative charge of which probably inhibits leakage back through the membrane. Chelation of intracellular Zn2+ as 'free Zn2+', or [Zn(OH2)6]2+, through the sulfonamide amido (after the loss of a proton) and quinolyl nitrogens occurs to give the [Zn(Zinquin A)(OH2)4]0 complex and subsequently [Zn(Zinquin A ) 2 ] 2 - . I n t h e l i k e l y e v e n t t h a t m o s t o f t h e intracellular Zn2+ is protein bound as [Zn(protein)]x, where the charge x varies with the nature of the protein, Zinquin A will either displace two of any waters bound in [Zn(protein)]x or protein binding groups to form [Zn(Zinquin A)(protein)](x-2). When irradiated at 358 nm [Zn(Zinquin A)(OH2)4]0 emits a blue fluorescence with a maximum at 490 nm and [Zn(Zinquin A)2]2- and [Zn(Zinquin A)(protein)](x-2) emit blue fluorescence with similar wavelength maxima. However, the fluorescence intensity, or quantum yield, of Zinquin A varies to some extent with its Zn2+-bound environment and thus gives only a semi-quantitative measure of [Zn2+] in the presence o f o t h e r Z n 2 +- b i n d i n g s p e c i e s ( 3 ) . A s i m i l a r reservation applies to most other Zn2+ sensors. Even so, Zinquin E/A has been used successfully to visualise Zn2+ in a range of biological systems exemplified by hepatocytes, pancreatic islet cells and apoptosing cells (4-6).

The Origin of Zn2+ Sensor FluorescenceWhen in a ground state molecule an electron absorbs

a photon to go to a higher energy state. The resultant excited state's lifetime determines the extent to which fluorescence accompanies the return of the electron to the ground state. If the excited state is sufficiently long-lived for some of its energy to be dissipated into molecular vibrational modes, the excited electron will drop to a lower excited state energy level before emitting a photon (of lower energy than that which it absorbed initially) and returning to the ground state. As a consequence, the light wavelength absorbed is of shorter wavelength than that emitted which represents the fluorescence of the molecule. Often the lifetime of the excited state molecule is shortened so that little if any dissipation of energy is possible before the excited electron falls back to the ground state, emitting a photon of the same wavelength as that absorbed. Under these circumstances the molecule is weakly fluorescent at best and its fluorescence is said to be 'quenched'. If Zn2+ binds to such a molecule and disables the process which quenches its fluorescence, t h e m o l e c u l e w i l l s i g n a l t h e Z n 2 + b i n d i n g b y fluorescing and thereby act as a sensor. Thus, it is probable that in Zinquin E/A a weak hydrogen bonding occurs between the hydrogen attached to the sulphonamide nitrogen and the quinolyl nitrogen, and that excitation by irradiation at 358 nm produces an excited state in which the hydrogen transfers to the quinolyl nitrogen and weakly hydrogen bonds to the sulphonamide nitrogen (excited state proton transfer or ESPT (7)). The energy of this excited state is largely d i s s i p a t e d i n t o m o l e c u l a r v i b r a t i o n s s o t h a t fluorescence is quenched as Zinquin E/A returns to the ground state. Upon coordination by Zn2+, the sulphonamide hydrogen is displaced as Zn2+ binds both nitrogens in the formation of [Zn(Zinquin A)(OH2)4]0 and the other complexes in Fig. 1. This removes the ESPT quenching pathway and the Zn2+-bound Zinquin A anion is fluorescent. This process is usually described as 'chelation enhancement of

fluorescence' or CEF. Several other quinoline based Zn2+ specific fluorescent sensors have been reported as have three other types of Zn2+ fluorescent sensors (1).

The Photo-Induced Electron Transfer (PET) Process and Fluorescence

A second type of Zn2+ fluorescent sensor also works by decreasing the quenching of fluorescence in a f l u o r o p h o r e t h r o u g h Z n 2 + b i n d i n g , b u t t h e photochemical mechanism and sensor design are different from that discussed above. The mechanism involves the interruption of a photo-induced electron transfer (PET) process as shown in Fig. 2. The sensor incorporates an electron donor site which binds Zn2+ (often an amine or other nitrogen donor group) and a fluorophore which is an electron acceptor. In the absence of Zn2+, photo-excitation of an electron of the fluorophore from the highest occupied molecular orbital (HOMO) to it lowest unoccupied molecular orbital (LUMO) occurs. This leaves an electronic vacancy in the flourophore HOMO, which is filled by transfer of an electron from the higher energy HOMO of the electron donor. The overall effect is that the e x c i t e d s t a t e l i f e t i m e i s s h o r t e n e d a n d l i t t l e fluorescence occurs. Upon binding Zn2+, the energy of the electron donor HOMO becomes less than that of the fluorophore HOMO so that photo-excitation of an electron from the fluorophore HOMO to the LUMO leads to a longer-lived excited state from which energy may be dissipated into vibrational modes, and thus fluorescence occurs. Typical of such sensors is ZnAF-2F DA which passes readily through cellular membranes and is subsequently deacylated by an esterase to give ZnAF-2F (Fig. 3). This binds Zn2+ through its nitrogen donor atoms whereupon the fluorescein-derived fluorophore fluoresces, as shown in a study of rat hippocampus (8). Another example is Zinpyr-4 (Fig. 3) which also complexes Zn2+ through its nitrogen atoms causing the fluorescein fluorophore to fluoresce, as shown by a study of seizure-damaged rat neurons (9).

SHOWCASE ON RESEARCH

Page 14 AUSTRALIAN BIOCHEMIST Vol 35 No 3 December 2004

Fig. 1. The operation of the Zinquin E/A Zn2+-selective fluorescent sensor.

Fluorescent Sensors of Zinc (II)in Biological Systems

SHOWCASE ON RESEARCH

The Photo-Induced Charge Transfer (PCT) Process and Fluorescence

A third type of sensor is the photo-induced charge transfer (PCT) sensor in which the fluorophore is fluorescent and contains an electron donating group conjugated with an electron acceptor group such that internal charge transfer from the donor to the acceptor occurs. When Zn2+ binds to the electron donor group, the light absorbance wavelength decreases (blue shift), as does the fluorescence wavelength, because the energy gap between the HOMO and LUMO orbitals, or S0 and S1 states, increases as a consequence of a decreased donation of electrons into the conjugated π system of the fluorophore. Conversely, if Zn2+ binds to the electron acceptor group, the HOMO/LUMO energy gap will decrease as the electron withdrawing c h a r a c t e r o f t h e a c c e p t o r i n c r e a s e s a n d t h e wavelengths of both absorption and fluorescence will increase (red shift). In practice, the shift in the fluorescence wavelength is often less than that in the absorption wavelength. Such PCT sensors are typified by ZnAF-R1 and ZnAF-R2 (Fig. 3) in which the fluorescence of the benzofuran-based fluorophore is not quenched by the amine group (10). Upon Zn2+ binding by the four nitrogen atoms, a blue shift occurs for the absorption wavelength with a lesser shift for the fluorescence wavelength. Somewhat ironically, fluorescent sensors designed to be Mg2+ and Ca2+ selective are often even more selective for Zn2+ and can only be used for detection of Mg2+ and Ca2+ in the presence of a screening agent such as TPEN (Fig. 3), which tightly binds Zn2+ through all six nitrogen atoms but has a weak affinity for Mg2+ and Ca2+. Thus, Indo-1 (Fig. 3) has a hundred-fold greater affinity for Zn2+ than for Ca2+ (11).

The Fluorescence Resonance Energy Transfer (FRET) Process and FluorescenceA fourth type of sensor is that in which energy

transfer between two adjacent fluorophores in the same molecule occurs; a process referred to as fluorescence resonance energy transfer (FRET). Here the shorter wavelength absorbing fluorophore is excited and emits a longer wavelength fluorescence which excites the longer wavelength absorbing fluorophore which then fluoresces at a yet longer wavelength. The sensor also incorporates a Zn2+ binding site and the binding of Zn2+ brings the two fluorophores closer together which enhances FRET and fluorescence of the second fluorophore to signal Zn2+ binding. Such a sensor is exemplified by a peptide-based sensor (Fig. 3) in which the fluorescein-derived fluorophore is excited at 430 nm, fluoresces at 521 nm and excites the lissamine-derived fluorophore which fluoresces at 596 nm (12). The binding of Zn2+ by the peptide backbone, based on the zinc-finger consensus sequence, shortens the distance between the fluorophores such that FRET and the fluorescence of the lissamine-derived fluorophore is enhanced to signal the binding of Zn2+. This sensor is sensitive to oxygen, as is the case with several of the more complex fluorophores, which limits their utility. Nevertheless several interesting Zn2+ sensors have been reported where carbonic anhydrase, a well known Zn2+ binding protein, has been modified to act as a Zn2+ sensor through the attachment of two fluorophores (14).

Vol 35 No 3 December 2004 AUSTRALIAN BIOCHEMIST Page 15

Fluorescent Sensors of Zinc (II)in Biological Systems

Fig. 2. The operation of a PET Zn2+-selective fluorescent sensor.Sometimes a spacer is present between the fluorophore and the donor group, and sometimes not.

ConclusionIt is clear from this brief survey that a plethora of Zn2+ sensors have been both developed and deployed in cellular studies. The majority of them are not generally available and require a specialist chemical understanding in their use. The consequence is that Zinquin E/A and the closely related TSQ (Fig. 3) are the most immediately usable Zn2+ fluorescent sensors presently available.

References1. Jiang, P. and Guo, Z. (2004) Coord. Chem.

Rev. 248, 205-2292. Hendrickson, K.M., Rodopoulos, T., Pittet,

P.-A., Mahadevan, I., Lincoln, S.F., Ward, A.D., Kurucsev, T., Duckworth, P.A., Forbes, I.J., Zalewski, P. and Betts, W.H. (1997) J. Chem. Soc., Dalton Trans. 3879-3882

3. Hendrickson, K.M., Geue, J.P., Wyness, O., Lincoln, S.F. and Ward, A.D. (2003) J. Am. Chem. Soc. 125, 3889-3895

4. Zalewski, P.D., Millard, S.H., Forbes, I.J., Kapaniris, O., Slavotinek, A., Betts, W.H., Ward, A.D., Lincoln, S.F. and Mahadevan, I. (1984) J. Histochem. Cytochem. 42, 877-884

5. Coyle, P., Zalewski, P.D., Philcox, J.C., Forbes, I.J., Ward, A.D., Lincoln, S.F., Mahadevan, I. and Rofe, A.M. (1994) Biochem. J. 303, 781-786

6. Zalewski, P.D., Forbes, I.J., Seamark, R.F., Borlinghaus, R., Betts, W.H., Lincoln, S.F. and Ward, A.D. (1994) Chem. Biol. 1, 153-161

7. Bardez, E., Devol, I., Larrey, B. and Valeur, B. (1997) J. Phys. Chem. B. 101, 7786-7793

8. Hirano, T., Kikuchi, K., Urano, Y. and Nagano, T. (2002) J. Am. Chem. Soc. 124, 6555-6562

9. Burdette, S.C., Frederickson, C.J., Bu, W. and Lippard, S.J. (2003) J. Am. Chem. Soc. 125, 1778-1787

10.Maruyama, S., Kikuchi, K., Urano, Y., Higuchi, T. and Nagano, T. (2002) J. Am. Chem. Soc. 124, 10650-10651

11.Jefferson, J.R., Hunt, J.B. and Ginsburg, A. (1990) Anal. Biochem. 187, 328-336

12.Godwin, H.A. and Berg, J.M. (1996) J. Am. Chem. Soc. 118, 6514-6515

13.Thompson, R.B. and Patchan, M.W. (1995) Anal. Biochem. 227,123-128

14.Thompson, R.B., Maliwal, B.P., Feliccia, V.L., Fierke, C.A. and McCall, K. (1998) Anal. Chem. 70, 4717-4723

SHOWCASE ON RESEARCH

Page 16 AUSTRALIAN BIOCHEMIST Vol 35 No 3 December 2004

Fluorescent Sensors of Zinc (II)in Biological Systems

Fig. 3. Some Zn2+-selective fluorescent sensors.