Recent development of zinc-fluorophores Eiichi Kimura† and Tohru Koike Department of Medicinal Chemistry School of Medicine Hiroshima University Minami-ku Hiroshima 734-8551 Japan Zinc-fluorophores have recently been attracting much interest in biological and environmental applications. How to detect selectively trace Zn2+ with efficient signal transduction is the central problem. Following carbonic anhydrasebased biosensors with fluorescent aromatic sulfonamides a chemosensor Zinquin is now extensively used to study the role of intracellular Zn2+ in cellular biology. New types of zinc-fluorophores zinc-finger peptides attached with fluorescent dyes and a dansylamide-pendant macrocyclic polyamine have been developed in 1996. The principles properties and limitations of these are discussed.1 Introduction to fluorescent indicators Quantitative analysis of trace metal ions with a selective analytical reagent has become extremely important for environmental and biological applications.1 Remarkable development of fluorescent indicators has been made for biologically important divalent metal ions in particular Ca2+ and Mg2+ with several selective fluorophores such as Fura-2 (1) Quin-2 (2) and Mag-indo-1 (3).2,3 d The criteria for these sensors are (i) stability (ii) metal selectivity (iii) metal affinity (iv) signal transduction (v) fluorescent signaling (vi) kinetically rapid sensitization (vii) ease of delivery to target systems and (viii) availability. For measurement of the dynamic mechanism of intracellular Ca2+ the typical concentration in resting cells is 50–200 nm and the intracellular physiological range is 10 nm–10 mm.Therefore as for metal affinity Ca2+-selective biosensors should possess a K (dissociation constant) near the median concentration (ca. 10–6 m). When a normal median concentration (given above) gives a 50% signal one could most effectively detect both concentration increases and concentration decreases. Fura-2 and Quin-2 in this regard are quite appropriate probes for the measurement † E-mail ekimura@ipc.hiroshima-u.ac.jp Eiichi Kimura obtained his BSc and MSc from the University of Tokyo and his PhD from the University of North Carolina under Professor James P. Collman in 1967. After postdoctoral years at Syntex and Chicago University he became an associate professor at Hiroshima University in 1970 where he is presently a professor.His research interests include the supramolecular chemistry of macrocyclic polyamines and their use in molecular recognition and as zinc-enzyme models. He was given the 2nd Izatt- Christensen award for macrocyclic chemistry in 1992. Tohru Koike Eiichi Kimura N ydrase Hydropho Environm O2S –NH Zn2+ N N NH HN N Em 468n Ex 320 n NH of intracellular Ca2+ concentrations.3 As for the desirable fluorescent signaling properties these are (a) intense fluorescence (b) excitation wavelengths exceeding 340 nm (to pass through glass microscope objectives and minimize UV-induced cell damage) with a wavelength corresponding to available laser sources and (c) emission wavelengths should shift by > 80 nm before and after complexation so that ratiometric titration can be utilized (for quantification) rather than mere intensity changes.Fura-2 for example fits these criteria. 2 History of classical zinc-fluorophores The zinc(ii) ion has been recognized as an important cation in biological systems (e.g. influencing DNA synthesis apoptosis gene expression and protein structure and function).4 The zinc(ii) ion is also implicated in the formation of amyloid plaques during the onset of Alzheimer’s disease. The relative concentration of free Zn2+ within biological cells varies from about 1 nm in the cytoplasm of many cells to about 1 mm in some vesicles.Clearly a mechanism must be available for moving Zn2+ ions into complexing sites and for pumping it to elevated concentrations that allow triggering mechanisms to operate as with Ca2+. The need for useful zinc-fluorophores to quantify trace Zn2+ ions is becoming more acute. The first zinc-fluorophore TSQ (4)5 was used as an histochemical stain for Zn2+ in various tissue sections of the brain heart and some other tissues. This stain was the only useful Zn2+-specific fluorophore that worked in the presence of physiological concentrations of Ca2+ and Mg2+. The complex of TSQ with free Zn2+ apparently has a stoichiometry of 2 1 TSQ/ Zn2+ but a 1 1 complex may equilibrate with protein-bound Zn2+. These TSQ-Zn2+ complexes were not fully identified nor fully characterized because of their complex structures and their stability constants were not determined.The fluorescence intensity (i.e. quantum yield) of the complex(es) varies with the media. Accordingly TSQ was far from an ideal fluorophore for the quantitative analysis of Zn2+. Tohru Koike was born in Hiroshima in 1959. After receiving his PhD in 1986 from Hiroshima University under the direction of Professor Eiichi Kimura he became an associate professor at Hiroshima University. His research interest is bioinorganic chemistry. He has been inventing new macrocyclic polyamines to disclose the intrinsic properties of Zn2+ in metalloproteins. 179 Chemical Society Reviews 1998 volume 27 COO– H3CO O COO– N N O COO– N COO– H3C O N O N COO– O COO– COO– N H3C COO– COO– 2 1 Quin-2 Fula-2 Kd(Ca2+) 60 nM Em 495 nm Ex 333 nm Kd(Ca2+) 145 nM Em 505 nm Ex 335 nm COO– COO– N COO– O 3 Mag-indo-1 NH Kd(Mg2+) 2.7 mM Em 417 nm Ex 330 nm COO– Whilst the TSQ-Zn2+ complexes were still chemically to be characterized a modified TSQ Zinquin 5 was developed and extensively used for cellular physiological studies by Zalewsky’s group.6,7 This was the first probe to visualize intracellular Zn2+ ions in living cells.An ester group is incorporated in 5 so that after the neutral lipophilic probe permeates into the cell the ester is hydrolyzed by intracellular esterases to become a carboxylate anionic form 6 and therefore be retained for a long time within the cell (see Scheme 1).Thus Zinquin became the first practical zinc-fluorophore to be used to determine the role of Zn2+ in the regulation of cell growth. Zinquin 5 can monitor loosely bound labile intracellular Zn2+ (not tightly bound Zn2+ in zinc-enzymes or zinc-finger proteins) by fluorescence video image analysis or fluorometric spectroscopy. The importance of cellular Zn2+ distribution in the process of apoptosis was first revealed by Zinquin for in zincrich cells such as hepatocytes and pancreatic islet b-cells the fluorescence is very intense. By fluorometric titration Zinquin was found to form both 1 1 and 2 1 complexes with Zn2+ with binding constants of 7.0 3 106 m21 and 11.7 3 106 m21 at pH 7.4.6 However the structure of these complexes was not determined but on the basis of our following findings 7 is a reasonable formula for the 1 1 complex where the sulfonamide is deprotonated.It is certain however that the stability constants are not big enough to permit interaction of Zinquin with the tightly bound (Kd << 1 nm) Zn2+ in metalloenzymes or zinc-finger proteins. The H3CO N HN TSQ Em 495nm Ex 334 nm CH3 SO2 4 Chemical Society Reviews 1998 volume 27 180 COO– COOEt O O Intracellular Esterases N NH N NH O2S O2S CH3 CH3 H3C H3C 5 Zinquin – H+ COO– N O2S 6 + Zn2+ O N– Zn2+ CH3 etc. H3C 7 1:1 Complex Em 490nm Ex 370 nm Scheme 1 intracellular Zn2+ chelator N,N,NA,NA-tetrakis(2-pyridylmethyl) ethylendiamine (TPEN) which has a much higher affinity towards Zn2+ can mask the Zn2+-dependent Zinquin’s fluorescence.6 Very weak fluorescence (at 490 nm) of a 2 mm solution of Zinquin at pH 7.4 was increased at subnanomolar concentrations of free Zn2+ and was fully saturated at 1 mm Zn2+.5 Fluorescence was enhanced 20-fold by 1 mm Zn2+.None of the other biologically relevant metal ions (Ca2+ Mg2+ Cu2+ Fe2+ Fe3+ Mn2+ Co2+ etc.) affected the Zn2+- dependent fluorescence of Zinquin. However when it comes to quantitative analysis of Zn2+ either in living cells or in other environments Zinquin is still far from satisfactory due to the mixed complexes it forms with varying fluorescence intensity.3 Development of new zinc-fluorophores In 1940 Mann and Keilin reported the discovery that sulfonamides inhibit zinc-containing carbonic anhydrase (CA).8 Chen and Kernohan showed that bovine erythrocyte carbonic anhydrase interacts with equimolar dansylamide 8 to form a highly fluorescent complex 9 with a dissociation constant Kd of 2.5 3 1027 m at pH 7.4.9 For reference the Kd value for Zn2+ binding to apoCA is 4 3 10212 m. The fluorescence of free dansylamide in water has an emission peak at 580 nm with a quantum yield of only 5.5% but the CA-bound dansylamide shifted the emission maximum to 468 nm with a much higher quantum yield of 84% (excited at 326 nm). The large emission blue shift is rationalized by a well shielded and extremely hydrophobic binding site and in addition by the sulfonamide group losing a proton (to form SO2NH2) upon binding to CA (see Scheme 2).Thus dansylamide may serve as a good candidate for a fluorescent probe for CA or Zn2+ in the presence of apoCA. The pKa value of the free SO2NH2 group is 9.8 either in the ground state or in the excited state. Mere deprotonation of the free SO2NH2 group (in the absence of CA) shifted the emission peak from 580 to 540 nm and the quantum yield from 5.5% to 8.5%. These chemical principles were adopted to a CA-based fiber optic zinc-biosensor developed by Thompson and Jones in 1993.10 The concentration of Zn2+ ion is proportional to the ratio of fluorescence intensities (at 460/560 nm) at 10–1000 nm (with 1 mm apoCA and 10 mm dansylamide in pH 7.4 HEPES buffer).A special advantage with the CA-dansylamide system N Carbonic Anhydrase O2S –NH pH 7.4 O2S NH2 Zn2+ N 8 HN N Dansylamide Em 580nm Ex 320 nm Scheme 2 is that a large wavelength shift in fluorescence with or without Zn2+ in CA permits the ratio of emission at two different wavelengths to be correlated with the analytical level. This linear range interestingly corresponds to the zinc(ii) concentration range in the ocean. A fiber optic sensor constructed using this approach however showed the zinc-detection limit to be reduced by tenfold. For practical application to the measurment of environmental Zn2+ (e.g. in sea water) a problem is the reversibility.The dissociation rate of Zn2+ from CA is ca. 1028 s21 which is too slow to take continuous data. Another serious problem was fiber attenuation. Back in 1988 chelation-enhanced fluorescence (CHEF) was reported by Czarnik et al. with 9,10-bis(2,5-dimethyl-2,5-diazahexyl) anthracene 10 for Zn2+ in CH3CN.11 In 1990 10 was N N N N 10 N Zn2+ NH HN NH 12 extended to macrocyclic system 11.12,13 A large CHEF effect by Zn2+ (14.4-fold) and Cd2+ (nine-fold) was observed with 11 (n = 2) at pH 10 in aqueous solution where the metal-free ligand is almost entirely a monoprotonated species. In fluorescence titration of 11 (n = 2) (10 mm) with Zn2+ (0–20 mm) in pH 12 buffer (highly alkaline) where 11 (n = 2) is unprotonated the emission maximum at 416 nm (excited at 335 nm) increases linearly until almost 1 1 complexation (to 12).The reason why such a high pH was employed was to avoid the intrinsic competition between H+ and Zn2+ for 11 at lower pHs. The protonation(s) and metal complexation at the macrocyclic polyamine moiety commonly inhibit the quenching process by free nitrogen atoms e.g. the protonated ligand 11 (n = 2)·2H+ at pH 7 showed almost 120-fold larger fluorescence intensity N Hydrophobic Environment N NH Em 468nm Ex 320 nm NH 9 N NH n 11 n = 1–5 than that of the free ligand 11 (n = 2) at pH 12. Thus 11 (n = 2) can not be a practical zinc-fluorophore under normal pH conditions. A conceptually new zinc-fluorophore 13 was designed in 1996 by Imperiali et al.14 A peptide 13 containing a zinc-finger motif (a strong Zn2+-binding site Cys2/His2)15 attached to a N O2S NH YQ CQY CEKR ADSSNLKT HIKTK HS CH3CONH NH2 NH O 2/ HO O O N N+ O COO– SO3 – N Lissamine (L) O O SO2 HN 13 dansylamide residue was synthesized (i) for selective and efficient Zn2+-binding (Kd = 1.4 3 10210 m at pH 7)16 and (ii) to create a hydrophobic environment around the encapsulated dansylamide residue upon its Zn2+ complexation.The addition of Zn2+ (0.1–1 mm) to the peptide 13 (1.4 mm) in pH 7 HEPES buffer resulted in a linearly increased emission peak at 475 nm (excited at 333 nm). In the absence of Zn2+ the emission maximum was 560 nm. The presence of 0.5 M Na+ 50 mm Mg2+ and 100 mm Co2+ did not interfere in the Zn2+ analysis.The design presented here consists of a synthetic polypeptide (25 amino acids) template and covalently attached fluorescent reporter dansylamide that is sensitive to any metal-induced conformational changes of the supporting framework yet remote from the Zn2+-binding site. The enhanced emission is mostly due to the reporter being placed in a hydrophobic environment. Problems with the zinc-finger motif in addition to its synthetic availability were its susceptibility to air oxidation of the cysteine residues and to redox active metal ions such as Cu2+. This was despite its high affinity for Zn2+ ions. In application to the reductive environment of some cells this may not be problematic however in aerobic oxidative environmental analysis this zinc-fluorophore may not be suitable.An oxidatively robust peptidyl zinc-fluorophore was later synthesized by making the peptide substitution from Cys2/His2 to Cys/His3 at the Zn2+-binding site.16 However the zinc affinity dropped (Kd = 3 3 1029 m at pH 7) and the fluorescence response to Zn2+ became too small to be useful as a sensitive zinc-fluorophore. Another modification from Cys His2 to Cys/Asp/His2 yielded a zinc sensor with enhanced oxidative stability but with further weakened affinity (Kd = 6.5 3 1028 m at pH 7) although this one is responsive to a submicromolar to micromolar concentration of Zn2+ in the presence of redox active Cu2+ and Fe2+. Another type of zinc-fluorophore 14 having a zinc-finger motif (Cys2/His2) was designed in 1996 by Berg et al.17 The peptide was attached with two fluorescent dyes fluorescein (F) 14 Fluorescein (F) ATK CPE CGKSFSQ C SDLVK HQRT HTG COO– as the energy donor and lissamine (L) as the acceptor to visualize zinc binding.In the absence of Zn2+ ion the peptide is unfolded as shown for Imperiali’s peptides and the dyes are Chemical Society Reviews 1998 volume 27 181 relatively far apart (i.e. small intramolecular energy transfer occurs between F and L). Upon Zn2+-binding to the Cys2/His2 site the peptide folds to bring the two fluorophores closer together increasing the amount of intramolecular energy transfer. The Zn2+-binding to 14 (3.7 mm) at pH 7.1 was monitored by increasing fluorescence (ca.2.3-fold) at 596 nm (excitation at 430 nm) with an increase in [Zn2+] which provides the 1 1 and 2 1 peptide to Zn2+ complex formation. The complexation had to be carried out under a reductive atmosphere of 95% N2/5% H2 to avoid peptide oxidation however. 4 A novel biomimetic zinc-fluorophore While engaged in elucidation of the roles of Zn2+ ions in zinc enzymes [in particular carbonic anhydrase (CA)] by means of macrocyclic polyamine complexes {e.g. 15 with 1,5,9-triazacyclododecane ([12]aneN3) and 16 with 1,4,7,10-tetraazacyclododecane (cyclen)},18–23 we have discovered intrinsic acid properties of the Zn2+ ion. One of the most outstanding properties is a strong affinity for aromatic sulfonamides,21 as illustrated by the formation of the strong bonds between Zn2+ and deprotonated sulfonamide N2 anions at physiological pH.Thus for the first time a chemical model 15c was presented to account for aromatic sulfonamide anions being good ligands for Zn2+ ion at the active center of CA making them strong inhibitors (see Scheme 2). The zinc enzyme models 15 and 16 also form stable 1 1 complexes with deprotonated weak acids such as thymine derivatives (16c) and barbital (16d) in neutral aqueous solution,24 which result from the Zn2+-bound OH2 species generated with pKa values of 7.3 (for 15a to 15b + H+) and 7.9 (for 16a to 16b + H+)18 acting as bases to dissociate the X X Zn2+ HN Zn2+ NH NH HN NH HN NH O N– c; X = 16a; X = OH2 b; X = OH– O O 15a; X = OH2 b; X = OH– c; X = NR S O N H – R N– O d; X = O NH O acidic protons.The resulting conjugate bases strongly bind to Zn2+ which compensate for the unfavorable deprotonations at neutral pH. We further demonstrated that tosylamidopropyl[ 12]aneN3 17 yields a very stable four-coordinate tetrahedral zinc(ii) complex 18 under physiological pH (see Scheme 3) where the aromatic sulfonamide N2 anion strongly CH3 CH3 O O S O O S –N HN + Zn2+ Zn2+ –3H+ N H N N HN pH ~7 2H+ NH NH 18 17 Tosylamidopropyl[12]aneN3 Scheme 3 binds to a Zn2+ ion from the fourth coordination site. On the basis of these basic studies on a CA-model a dansylamide- Chemical Society Reviews 1998 volume 27 182 pendant macrocyclic tetraamine (dansylamidoethylcyclen) 1925 was designed for a new type of selective and efficient zincfluorophore 20.The reason why we initially adopted the macrocyclic tetraamine (L) is because it forms a much more stable Zn2+ complex (K = [ZnL]/[Zn2+][L] = 1015.3 m21)26 than [12]aneN3 (K = 108.4 m21)18 in H2O at 25 °C. 4.1 Synthesis of dansylamidoethylcyclen Dansylamidoethylcyclen 19 was initially synthesized according to Scheme 4,25 but recently a more convenient synthetic route has been developed as shown in Scheme 5 which is used commercially.27An X-ray crystal structure of the Zn2+ complex 20 confirmed the 5-coordinate disordered square-pyramid structure with a short Zn2+-sulfonamide N2 bonding (1.97 Å).25 O S O NH N NH HN NH HN O NH N HN 4.2 Zinc(ii) affinity of dansylamidoethylcyclen The zinc(ii) complexation equilibria were determined by potentiometric pH titration of dansylamidoethylcyclen 19 in the presence of an equimolar amount of Zn2+ at 25 °C with I = 0.10 (NaNO3).The complexation constant for 19 (HL) (K = [20]/[L2][Zn2+]) was establised to be 1020.8 m21. The dissociation constants Kd at pH 7.8 were calculated from the complexation constants as summarized with reference values in Table 1. It is of interest to note that the Kd values are adjustable within physiological pH (e.g. 1.4 3 10210 m at pH 7.0 to 5.5 3 10213 m at pH 7.8) for the new biomimetic zinc-fluorophore 19. As the pH is raised the K 20 d value for 19 becomes smaller than the HN N BH3•THF HN N NH HN 19 Dansylamidoethylcyclen HN H2N Zn2+ i) dansyl chloride K2CO3 ii) NaClO4 in H2O pH 7 20•ClO4 – N N O S O -N HN Zn2+ N NH HN O O O BrCH2CN NH HN NH in MeOH CN in THF i) Amberlite IRA-400 NH HN ii) Zn(ClO4)2 in EtOH 5HCl 2ClO4 – NH2 i) 5 equiv.EDTA 19•5HCl ii) 6 M HCl Scheme 4 N HN Boc Boc N N + N O2S Cl Boc N O2S NH NH HCl aq in MeOH N Boc N N Boc Cyclen Boc = tert-butoxycarbonyl KI/K2CO3 in CH3CN [12]aneN3 1.031024 19•5HCl Zinquin (1 1 complex)a 1.431027 N 4.4310211 a Boc Scheme 5 Table 1 Comparison of dissociation constants Kd (M) ( = [Zn2+][zinc(ii) complex]/[free ligand]) at 25 °C and pH 7.8 19 5.5310213 Calculated using the 1 1 complexation constant (at pH 7.4) given in ref.6. Kd values for the zinc-finger consensus peptides (e.g. 5.7 3 10212 m at pH 7.0).15 The distribution of various species in solution is shown as a function of pH at [total Zn2+] = [total ligand] = 1 mm in Fig. 1 for 19. Most remarkably 19 (almost in HL·2H+ form 1 mm) sequesters nearly 100% of trace Zn2+ (1 mm) in the form of stoichiometric ZnL 20 at physiological pH of 7.8. Such a strong affinity to Zn2+ is one of the most characteristic properties of the new macrocyclic ligand [in comparison to Zinquin 5 and anthracene-pendant cyclen 11 (n = 2)] and will be very useful for quantifying trace amounts of Zn2+ in environmental and biological systems.4.3 Fluorescent signalling behaviour of dansylamidoethylcyclen In principle a direct signal transduction linked with Zn2+ sensing is straightforward and should be more desirable than indirect ones (such as the metal-induced conformational changes).15,16 Thus the use of dansylamide for both Zn2+ recognition and fluorescence signaling would make a simpler and side-effects-free probe. Despite similar UV excitation spectra the fluorescence emission spectra vary dramatically for protonated ligand 19·2H+ (HL·2H+) at pH 7.8 deprotonated ligand (L2) at pH 12.8 Zn2+ complex 20 (ZnL) at pH 7.8 and Cu2+ complex with 19 (CuL) at pH 7.8 (see Fig. 2).25 While the non-metallated dansylamide deprotonation of HL·2H+ to L2 without Zn2+ at high pH brought about only ca.20% increase in the emission intensity the dansylamide deprotonation with Zn2+ at pH 7.8 increased the emission intensity by 4.9-fold at 540 nm and ten-fold at 490 nm. In contrast the dansylamide Fig. 1. Distribution diagram for the zinc(ii) species in 1 mm 19/1 mm Zn2+ system as a function of pH at 25 °C where ZnHL is a monoprotonated species of 20. Fig. 2 UV absorption spectra at 25 °C and pH 7.3 (10 mm HEPES) with I = 0.1 (NaNO3) (a) 0.1 mm 19·2H+ (HL·2H+) lmax = 330 nm; (b) 0.1 mm 20 (ZnL) lmax = 323 nm; (c) 0.1 mm copper(ii) complex of 19 (CuL) lmax = 305 nm. Fluorescence spectra by 330 nm excitation at 25 °C with I = 0.1 (NaNO3); (d) 10 mm HL·2H+ at pH 7.8 (10 mm EPPS buffer) lmax = 582 nm; (e) 10 mm L2 at pH 12.8 lmax = 578 nm; (f) 10 mm ZnL at pH 7.8 (10 mm EPPS buffer) lmax = 540 nm.10 mm CuL has no fluorescence under the same conditions. deprotonation with Cu2+ completely quenched the fluorescence. The fluorescence maximum of HL·2H+ (582 nm) at neutral pH blue-shifted upon zinc(ii) complexation (ZnL) to 540 nm. A greater fluorescence blue shift (580 nm to 468 nm) and intensity enhancement (15.3-fold in quantum yield excited at 320 nm) were reported for the dansylamide complexation with carbonic anhydrase (CA),9 which were accounted for by the hydrophobic environment and the deprotonation of dansylamide on Zn2+ at the active center of CA. In support of this explanation the fluorescence maximum (540 nm) of 20 in H2O moved toward shorter wavelength in organic solvents with higher quantum yields 502 nm in MeOH and 490 nm in CH3CN.25,27 4.4 Evaluation of the zinc sensitivity of dansylamidoethylcyclen The fluorescence changes of 19 (5 mm) with various metal ions (5 mm) at pH 7.3 (HEPES buffer) and 25 °C are summarized in Fig.3.25,28 The addition of various concentrations of Zn2+ ion (0–10 mm) resulted in increased emission upon excitation at 330 nm as shown in Fig. 4. The response (at 528 nm) was linear between 0.1 and 5 mm until it reached a 1 1 [19]/[Zn2+] ratio and then became a plateau. These responses indicate that the increase in fluorescence is stoichiometric due totally to the 1 1 ZnL formation and moreover ZnL is so stable that even at 183 Chemical Society Reviews 1998 volume 27 Fig.3 Comparison of the relative fluorescence intensity of 5 mm 19 in the presence of various additives at 25 °C and pH 7.3 (1 mm HEPES) with I = 0.1 (NaNO3). The data marked with * were collected without the supporting electrolyte. nanomolar concentrations it does not dissociate which is in good agreement with the results obtained by the potentiometric pH titration (see the pH distribution curve in Fig. 1). The Zn2+- dependent fluorescence was unaffected by the presence of an excess amount of Zn2+. On the other hand Cu2+ ion linearly diminished the fluorescence emission until complete quenching at [19]/[Cu2+] = 1 (see Fig. 4). Other fluorescence-quenching metal ions (paramagnetic Co2+ easily reducible Hg2+ Pb2+) that tend to bind fairly stongly with cyclen also caused minor intramolecular quenching although the effects were not so drastic as Cu2+.Cu2+ ion forms the most stable five-coordinate complex CuL (K = [CuL]/[Cu2+][L–] > 1030 m21 at 25 °C),25 which account for the most dramatic effect by Cu2+. When the binding to cyclen is not strong (in aqueous solution) as demonstrated by Fe2+ Fe3+ Mn2+ or Mg2+ there was no effect on the fluorescence. The fluorescence intensity of 5 mm ZnL 20 was practically unaffected by the presence of physiological concentrations ( > 103-fold) of Na+ K+ Ca2+ or Mg2+ at pH 7.3. Fig. 4 Fluorescence emission response (at 528 nm) of 5 mm 19 to increasing levels of Zn2+ or Cu2+ at 25 °C and pH 7.3 (1 mm HEPES) with I = 0.1 (NaNO3).The interference of Cu2+ in the fluorescence response of 20 by Cu2+ could be prevented by using bovine serum albumin (BSA) which specifically masked Cu2+.28 The mixture of [Zn2+] = [Cu2+] = [19] = 5 mm at pH 7.3 showed no fluorescence. However with an increasing amount of BSA the fluorescence increased due to 20 and at [BSA] = 50 mm the original intensity (without Cu2+) was regained (see Fig. 4). Chemical Society Reviews 1998 volume 27 184 5 Future perspectives on zinc-fluorophores Currently the macrocyclic polyamine attached with a dansylamide-pendant 19 seems to offer the most useful prototype for practical zinc-fluorophores with the various criteria described in the introduction (i) the ligands are easy to make and very robust; (ii) the affinity for Zn2+ is extremely high catching trace Zn2+ in nanomolar concentrations at physiological pH; (iii) the Zn2+ selectivity in fluorescence sensing is very high and the zinc-fluorescence perturbing metal ions are limited (e.g.Hg2+ Pb2+); (iv) the most interfering metal Cu2+ can be masked by use of BSA. Further studies to be made for biological applications are (i) kinetic aspects (e.g. how fast is the Zn2+ sensitization) (ii) delivery into cells and how long it remains responsive to intracellular Zn2+ and (iii) improvement in the fluorescence efficiency. It is anticipated that these problems may be solved by appropriate modification of the relatively simple macrocyclic structure. 6 References 1 A. W. Czarnik Chem.Biol. 1995 2 423. 2 G. Grynkiewicz M. Poenie and R. Y. Ysien J. Biol. Chem. 1985 260 3440. 3 R. P. Haugland Handbook of Fluorescent Probes and Research Chemicals ed. by M. T. Z. Spence Molecular Probes Eugene 6th edn. 4 J. J. Fra�usto da Silva and R. J. P. Williams The Biological Chemistry of 5 C. J. Frederickson E. J. Kasarskis D. Ringo and R. E. Frederickson J. 6 P. D. Zalewski I. J. Forbes and W. H. Betts Biochem. J. 1993 296 7 P. D. Zalewski I. J. Forbes R. F. Seamark R. Borlinghaus W. H. Betts 1996 p. 503. the Elements Clarendon Press Oxford 1991 p. 302. Neurosci. Methods 1987 20 91. 403. S. F. Lincoln and A. D. Ward Chem. Biol. 1994 3 153. 8 T. Mann and D. Keilin Nature 1940 146 164. 9 R. F. Chen and J. C. Kernohan J. Biol. Chem. 1967 247 5813.10 R. B. Thompson and E. R. Jones Anal. Chem. 1993 65 730. 11 M. H. Huston K. W. Haider and A. W. Czarnik J. Am. Chem. Soc. 1988 110 4460. 12 E. U. Akkaya M. H. Huston and A. W. Czarnik J. Am. Chem. Soc. 1990 112 3590. 13 A. W. Czarnik Acc. Chem. Res. 1994 27 302. 14 G. K. Walkup and B. Imperiali J. Am. Chem. Soc. 1996 118 3053. 15 B. A. Krizek D. L. Merkle and J. M. Berg Inorg. Chem. 1993 32 937. 16 G. K. Walkup and B. Imperiali J. Am. Chem. Soc. 1997 119 3443. 17 H. A. Godwin and J. M. Berg J. Am. Chem. Soc. 1996 118 6514. 18 E. Kimura T. Shiota T. Koike M. Shiro and M. Kodama J. Am. Chem. Soc. 1990 112 5805. 19 T. Koike and E. Kimura J. Am. Chem. Soc. 1991 113 8935. 20 E. Kimura T. Koike M. Shionoya and M. Shiro Chem. Lett. 1992 787. 21 T. Koike E. Kimura I. Nakamura Y. Hashimoto and M. Shiro J. Am. Chem. Soc. 1992 114 7338. 22 X. Zhang R. van Eldik T. Koike and E. Kimura Inorg. Chem. 1993 32 5749. 23 T. Koike M. Takamura and E. Kimura J. Am. Chem. Soc. 1994 116 8443. 24 T. Koike M. Takashige E. Kimura H. Fujioka and M. Shiro Chem. Eur. J. 1996 2 617. 25 T. Koike T. Watanabe S. Aoki E. Kimura and M. Shiro J. Am. Chem. Soc. 1996 118 12696. The fluorescence is so sensitive that extreme care to exclude contamination should be exercised in preparing the analytical solution (e.g. our experience showed that for the supporting electrolyte optical grade purity was needed for micromolar fluorometric analysis). 26 T. Koike S. Kajitani I. Nakamura E. Kimura and M. Shiro J. Am. Chem. Soc. 1995 117 1210. 27 K. Takesako Dojin News 1998 86 18. Dansylamidoethylcyclen 19·5HCl is commercially available from Funakoshi Ltd. (http:/ /www.funakoshi.co.jp/) and Dojindo (http://www.dojindo.co.jp/) in Japan. 28 E. Kimura S. Afr. J. Chem. 1997 50 in press. Received 2nd July 1997 Accepted 29th January 19