A ‘turn-on’ FRET peptide sensor based on the mercury bindingprotein MerP

Abstract

A new fluorescent peptidyl chemosensor based on the mercury binding MerP protein with fluorescence resonance energy transfer (FRET) capabilities has been synthesized via Fmoc solid-phase peptide synthesis. The metal chelating unit, which is flanked by the fluorophores tryptophan (donor) and dansyl (acceptor), contains amino acids from MerP's metal binding loop (sequence: dansyl-Gly-Gly-Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys-Gly-Gly-Trp-CONH₂). A FRET enhancement or ‘turn-on’ response was observed for Hg²⁺ as well as for Zn²⁺, Cd²⁺ and Ag⁺ in a pure aqueous solution at pH 7.0. The emission intensity of the acceptor was used to monitor the concentration of these metals ions with detection limits of 280, 6, 103 and 496 µg L⁻¹, respectively. No response was observed for the other transition, alkali and alkaline earth metals tested. The fluorescent enhancement observed is unique for Hg²⁺ since this metal generally quenches fluorescence . The acceptorfluorescence increase resulting from metal binding-induced FRET suggests a sensor that is inherently more sensitive than one based on quenching by the binding event.

---

Introduction

Determination of low level concentrations of heavy metal ions has become significant due to the severe risks they pose for human health and the environment.¹ This has prompted research into the development of fluorescent chemosensors for their detection in environmental and biological samples.^2–4 These chemosensors are typically composed of two covalently linked structural subunits: a fluorophore (for signal transduction) and an ionophore (for selective recognition of the metal ion). When designing these sensors, intense effort is put into maximizing the selectivity of the metal chelating unit. Chelating units composed of organic molecules have been used, but synthesis is rigorous and binding is not always reversible.^3,5–15 Peptide motifs prove to be a viable alternative since they exhibit metal selectivity, can be easily synthesized via 9-fluorenyl-methoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS)^16,17 and are usable in aqueous solutions.

In this study a peptide fragment based on the mercury bindingprotein MerP was used as the metal chelating unit. MerP is a member of the bacterial mercury detoxification system^18,19 and is responsible for binding Hg²⁺ in the periplasm and transferring it to transport protein MerT. Like other metal bindingproteins , MerP contains the Cys-X-X-Cys motif and coordination to the cysteines is the dominant metal binding mechanism.²⁰ While the full protein is 72 amino acids in length, studies have shown that the 18 residue fragment Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys from the metal binding loop has structural and binding characteristics similar to the full protein .^21,22

Typically, metal binding is detected by the quenching of a single fluorophore, e.g.dansyl,^{5,9,12,23–27} lucifer yellow,²⁸anthracene.^6,29–31 Although concentration-dependent quenching mechanisms have proven successful, it is inherently less sensitive than methods that producefluorescence or ‘turn-on’ as a result of binding.³² Also, it is often difficult to distinguish analyte response from sensor degradation when quenching is relied upon for quantitation.

The current study utilizes fluorescence resonance energy transfer (FRET) as a mechanism of detecting metal binding, allowing fluorescence enhancement to be monitored. Conceptually, if the chelating unit folds around the metal as it binds, the fluorophores may be brought closer together, causing increased transfer of energy from the donor fluorophore to the acceptor fluorophore. This has been successfully demonstrated by Imperiali and co-workers for Ni²⁺,³³ and Godwin and Berg for Zn²⁺.³⁴ In comparison to conventional fluorescence , FRET allows a larger wavelength separation between the excitation and emission wavelengths, thereby permitting the use of lower resolution wavelength isolation devices (e.g. filters) to measure emission without interference from the excitation source.

This paper reports on the synthesis of a new peptidyl chemosensor with FRET capabilities based on the mercury bindingprotein MerP that can be operated in aqueous solution at pH 7.0. The 23 residue peptide (sequence: dansyl-Gly-Gly-Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys-Gly-Gly-Trp-CONH₂) contains amino acids from MerP's metal binding loop. The FRET pair (tryptophan as donor and dansyl as acceptor) was conveniently attached during SPPS and are separated from the metal chelating unit by two glycine residues. Although MerP (and its metal binding loop fragment) has some binding affinity for other metals, the structures of the metal-bound forms are very different. While the protein forms a loop around the bound mercury, this loop is distorted or even non-existent with other metals.²² Because FRET is a distance-dependent interaction, the structural differences are exploited by this detection mechanism. Prior to this, there have been very few examples of Hg²⁺ chemosensors utilizing fluorescence enhancement due to mercury's propensity to quench fluorescence by enhanced spin–orbit coupling.³⁵ Of these chemosensors, most are only usable in organic^36–42 or mixed organic–aqueous solutions.^43–50 Only four could be operated in a pure aqueous solution.^51–54 Additionally, only one example of a FRET-based chemosensor for Hg²⁺ has been attempted, but a quenching mechanism dominated.⁵⁵

Experimental

Chemicals

All chemicals were reagent grade unless noted, and deionized distilled water was used to prepare solutions. Peptide synthesis reagents N-dansyl-N′-Fmoc-ethylenediamine-MPB-AM (Dansyl NovaTag^®) resin (100–200 mesh; 0.38 mmol g⁻¹), Wang resin (100–200 mesh, 1.2 mmol g⁻¹), glycine (Fmoc-Gly-OH), threonine [Fmoc-Thr(t-butyl ester (OtBu))-OH], leucine (Fmoc-Leu-OH), alanine (Fmoc-Ala-OH), valine (Fmoc-Val-OH), proline (Fmoc-Pro-OH), cysteine [Fmoc-Cys(Trt)-OH], isoleucine (Fmoc-Ile-OH), lysine [Fmoc-Lys(Boc)-OH], tryptophan [Fmoc-Trp(Boc)-OH] and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) were used as received from Novabiochem. All the amino acids were of L-configuration. Metal-containing solutions were prepared by dilution from 1000 µg ml⁻¹ stock solutions. A 0.05 mol L⁻¹ [N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)] (HEPES) (Aldrich) buffer was prepared and adjusted to pH 7.0 with ammonium hydroxide (Fisher). Other reagents used include trifluoroacetic acid (99%) (TFA) (Acros), triisopropylsilane (99%) (TIPS) (Acros), ethyl ether (Fisher), (ethylenedinitrilo)tetraacetic acid (EDTA) (EM Science), dithiothreitol (DTT) (Acros), N-methylmorpholine (NMM) (Fisher), N-methylpyrrolidone (NMP) (Fisher) and piperidine (99%) (Fisher).

Apparatus

A Photon Technologies International Quanta Master Spectrofluorimeter (model QM-4/2005) was used for all fluorescence measurements.

Peptide synthesis

A peptide consisting of the sequence dansyl-Gly-Gly-Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys-Gly-Gly-Trp-CONH₂ (P1) was synthesized on Dansyl NovaTag^® resin and the same peptide without dansyl (P2) was synthesized on Wang resin by Fmoc solid-phase peptide synthesis using a Ranin Symphony Quartet automated peptide synthesizer. Cleavage protocols have been described earlier.⁵⁶ The peptide masses (2494.3 for P1 and 2217.7 for P2) were confirmed using electrospray mass spectrometry and the purity of each sequence (63% for P1 and 73% for P2) was determined by reverse-phase HPLC . For both P1 and P2, no other single component was present in excess of 15%.

Fluorescence studies

Single-metal response studies. Fluorescence emission spectra were collected from 10 µmol L⁻¹ solutions of P1 and P2 (pH 7.0, 50 mmol L⁻¹HEPES, 3 µmol L⁻¹DTT). Various DTT concentrations (1–10 µmol L⁻¹) were tested due to concerns of metal–DTT complexation, but no change in fluorescence response was observed. The response of P1 and P2 to various metal cations (Ag⁺, Ca²⁺, Cd²⁺, Hg²⁺, K⁺, Mg²⁺, Na⁺, Ni²⁺ and Zn²⁺) was determined by adding a range of metal concentrations (1–60 µmol L⁻¹) to each peptide solution from 0.002 mol L⁻¹ metal stock solutions. FRET studies (monitoring emission of Trp and dansyl) were conducted on P1 and single fluorophore studies (monitoring the emission of Trp) were conducted on P2. For the tryptophan emission, intensities at 348 nm were used. When determining the signal intensity for dansyl chloride emission, an average of intensities between 545 and 550 nm was used except for Hg²⁺, Cd²⁺, Zn²⁺and Ag⁺, where metal binding resulted in a blue shift of emission and the 498–510 nm range was used.

Multi-metal reponse studies. A 10 µmol L⁻¹ P1 solution (pH 7.0, 50 mmol L⁻¹HEPES, 3 µmol L⁻¹DTT) was prepared. A range of Cd²⁺, Hg²⁺ and Zn²⁺ concentrations (0.5–30 µmol L⁻¹) was added to the P1 solution from 0.002 mol L⁻¹ metal stock solutions to determine the response of P1 to different ratios of the metals.

FRET measurement. In order to determine the distance (r) between the fluorophores, eqn (1)⁵⁷ was used: where R₀ is the Forster distance and r is the acceptor–donor distance. For the measurement of the FRET efficiency (E), the emission of P2, which contains only the donortryptophan (I_D), was compared to the emission of P1, which contains both the donortryptophan and the acceptordansyl (I_DA). The FRET efficiency was calculated by:⁵⁷The Forster distance, R₀, for tryptophan/dansyl has been previously determined to be 21 Å⁵⁸ and their spectral overlap has been illustrated.^59,60

The distance between the fluorophores is ca. 71 Å if P1 is completely unfolded.

Results and discussion

Single-metal fluorescence response studies

For P1, the addition of Hg²⁺ resulted in a decrease in the tryptophan (donor) emission intensity and an increase in the dansyl emission intensity (Fig. 1a), indicating an increase in FRET due to Hg²⁺ binding. This response was reversible after the addition of excess EDTA. A ca. 2-fold increase (the ratio of P1 in the presence of Hg²⁺ to that in the absence of Hg²⁺) in dansyl emission was observed as well as a 35 nm blue shift in its emission to 510 nm. The blue shift in dansyl's emission is not unexpected as dansyl is an environmentally sensitive fluorophore.⁶¹ As peptide folding by metal binding occurs, the fluorophore is shielded from the polar solvent, causing a blue shift in its emission wavelength. Calibration curves were constructed from the fluorescence spectra to illustrate the quantitative dependence of the fluorescence on Hg²⁺ concentration. The detection limit for Hg²⁺ was found to be 280 µg L⁻¹. Although this is higher than the EPA's drinking water maximum contaminant level (MCL) of 2 µg L⁻¹,⁶² it is lower than^41,45,50 or comparable to⁶³ the detection limits of several existing Hg²⁺ fluorescent chemosensors in the literature. Additionally, this detection limit was achieved without any optimization techniques, i.e. a more intense light source, a longer integration time, etc. With optimization, this sensor could very well detect the level required by the EPA.

Fig. 1 Relationship of the fluorescence of P1 (50 mmol L⁻¹HEPES, pH 7.0) to the concentration of a) Hg²⁺, b) Zn²⁺, c) Cd²⁺ and d) Ag⁺. λ_excitation = 290 nm. All spectra were smoothed using Savitzky–Golay least squares smoothing routine with a 21-point window (Origin).

Addition of Cd²⁺, Zn²⁺and Ag⁺ to P1 also resulted in a metal binding-induced FRET response (Fig. 1b–d) and this response was reversible after the addition of excess EDTA. The largest FRET increase of any metal was observed for Zn²⁺, which caused a ca. 11-fold increase in dansyl emission along with a 47 nm blue shift of the peak emission to 498 nm. The detection limit for Zn²⁺ was found to be 6 µg L⁻¹, well below the EPA's drinking water MCL of 5 mg L⁻¹.⁶⁴Cd²⁺ addition resulted in the second largest FRET response. A ca. 6-fold increase in dansyl emission and a 45 nm blue shift to 500 nm was observed. The detection limit of Cd²⁺ was found to be 103 µg L⁻¹. While this is above the EPA drinking water MCL of 5 µg L⁻¹,⁶⁴ it is only the third example of a fluorescence sensor that utilizes enhancement for the detection of Cd²⁺. Additionally, the focus of the previous examples was not on metal detection, but rather on using enhancement to monitor protein conformational changes⁶⁵ and the synthesis of a dual receptor system for general sensing of anions and cations.⁶⁶ Similarly to Hg²⁺, Ag⁺ addition resulted in a ca. 2-fold increase in dansyl emission intensity. A 39 nm blue shift to 506 nm was also observed. The detection limit for Ag⁺ was found to be 496 µg L⁻¹. A summary of these results is shown in Table 1.

Table 1 Spectroscopic data for P1

Metal	λ emission/nm^a	Enhancement factor^b	R/Å^c	Detection limit/µg L^−1	Log K^d
a Dansyl emission peak after addition of metal. The P1 emission intensity for dansyl chloride in the absence of metal is 545 nm. b Ratio of the intensity of P1 in the presence of metal ion to that in the absence of metal ion. c The distance between the fluorophores (R) before the addition of metal is 21.0 ± 0.6 Å. d Metal binding constant was obtained via non-linear fitting of the fluorescence titration data. Although P1 contained more than one site for these metals, only one could be determined.
Hg^2+	510	2	19.4 ± 0.6	280	4.4
Zn^2+	498	11	16.6 ± 0.7	6	5.2
Cd^2+	500	6	17.9 ± 0.5	103	5.9
Ag^+	506	2	19.1 ± 0.6	496	5.4

---

Although this binding motif was taken from the mercury bindingprotein MerP, it is not surprising that it binds other metals in addition to Hg²⁺. The Cys-X-X-Cys motif present in MerP is also found in many other soft-metal bindingproteins including cadmium transportprotein CadA,⁶⁷ the zinc finger domains⁶⁸ and superoxide dismutase, a yeast copper- and zinc-transporting protein .⁶⁹ Additionally, studies conducted by Opella and co-workers on MerP's metal binding loop fragment showed affinity for Zn²⁺ (log K = 3.5), Cd²⁺ (log K = 3.4) and Ag⁺ (log K = 3.4), which are not significantly different than that found for Hg²⁺ (log K = 4.0).²² It is curious that this sensor does not have a larger response to Hg²⁺, but this may be due to the apparent quenching of the Trpdonor by Hg²⁺, which will be discussed further.

The fluorescence response of P1 for a number of metal ions is presented in Table 2. P1 had no response to other transition, alkali and alkaline earth metals tested, illustrating its potential use in a variety of matrices. In addition to selectivity, the robustness of the sensor was also evaluated by varying the pH. When the pH was lowered to 3.5, dansyl's emission intensity decreased noticeably by a factor of ca. 5, but was restored when the pH was returned to 7.0.

Table 2 Enhancement response of 10 µmol L⁻¹ P1 (50 mmol L⁻¹HEPES, pH 7.0) in the presence of various metal cations (30 µmol L⁻¹)

Metal ion	Enhancement factor^a
a Ratio of the intensity of P1 in the presence of metal ion to that in the absence of metal ion.
Ag^+	2
Ca^2+	0
Cd^2+	6
Hg^2+	2
K^+	0
Na^+	0
Ni^2+	−1
Mg^2+	0
Zn^2+	11

---

Evaluation of conditional stability constants

Fig. 2a–d shows that the maximum metal : peptide binding ratio is approximately 3 for Hg²⁺ and Zn²⁺ and 2 for Cd²⁺ and Ag⁺. Opella and co-workers previously reported a metal : peptide binding ratio of 1 for all of these metals.²² It is possible that the additional residues (four glycines and two fluorophores) in this study could allow for more binding sites or change the structure of the peptide such that other residues are available for binding.

Fig. 2 Fluorescent binding curves of P1 with a) Hg²⁺, b) Zn²⁺, b) Cd²⁺ and d) Ag⁺. λ_excitation = 290 nm. The metal to peptide binding ratio is 2 : 1 for Cd²⁺ and Ag⁺ and 3 : 1 for Hg²⁺ and Zn²⁺.

While the FRET signal increases with metal concentration in all cases, it would not be surprising that the acceptor-to-donor distances would be different depending on whether one or two metals were bound. Hence the fluorescence sensitivity could very likely change whether P1 is binding one, two or three cations. Thus, it is difficult to confidently state that the proportionality constant relating the signal to the amount of metal bound has a constant value. For example, looking at the response for Zn²⁺ (Fig. 2b), it is relatively obvious that binding the first metal (0–10 µM) produces a smaller FRET signal than when the second metal is bound (ca. 10–20 µM). Deconvoluting the changing response is difficult but necessary if one were to attempt to develop an isotherm from which to extract log K values. In contrast to Zn²⁺, Cd²⁺ binding (Fig. 2c) appears to be relatively well behaved in spite of the obvious 2 : 1 metal : peptide ratio at saturation.

In an attempt to get some binding information, the data in Fig. 2 were used to construct binding isotherms based on a one-site model. Using Graphpad Prism 4^®, conditional stability constants were calculated for each metal. Although P1 bound these metals in ratios greater than one, only one binding site log K was determined due, in part, to the shape of the binding curve. From a fit of the data, the estimated log K is 4.4 for Hg²⁺, 5.2 for Zn²⁺, 5.9 for Cd²⁺ and 5.4 for Ag⁺. Similarly to previous reports,²² the values for Zn²⁺, Cd²⁺ and Ag⁺ are not significantly different. While goodness-of-fit was poorest for Hg²⁺, an R² value of 0.999 was obtained for Cd²⁺ binding.

When looking at Table 1, it is interesting to note that these log K values do not match the amount of FRET enhancement. For instance, Zn²⁺ binding resulted in the largest enhancement, but its log K is smaller than Cd²⁺ and Ag⁺. This is not surprising since there is no assurance that stronger binding would necessarily bring the fluorophores in closer proximity. Additional structural studies to elucidate the conformation of the peptide with and without a particular metal will be needed to make a more definitive statement.

Determination of FRET efficiency

Using eqn (1), the FRET efficiency without metal was determined to be 49 ± 4%, which corresponds to a distance of only 21.0 ± 0.6 Å between the fluorophores, indicating that P1 (71 Å when completely elongated) is coiled or folded even before the addition of the metal.

Eqn (1) was also used to calculate the FRET efficiency and distance between the fluorophores for P1 after metal binding, and these values are reported in Table 1. As expected, a large change in distance was seen for Zn²⁺ and Cd²⁺ which had large FRET enhancement. Hg²⁺ and Ag⁺, which had the smallest enhancement, also had small distance changes. The small FRET efficiency increase for Hg²⁺ may be explained by its propensity to coordinate with amines .^70,71 When monitoring P2, which contains just the Trpdonor, its emission was quenched to 30.7% of its initial value at 30 µmol L⁻¹Hg²⁺. It has been shown that Hg²⁺ complexes with Trp's indole ring, causing quenching .⁷² If this occurs, the amount of energy that Trp is able to transfer to dansyl will yield an apparent reduction in the FRET signal. Additionally, Hg²⁺ binding to other amine -containing sites may prevent the peptide from obtaining the loop conformation described by Opella and co-workers,²² which could also result in a lower FRET efficiency. It should be noted that no quenching of dansyl by Hg²⁺ was observed when P1 was excited only at dansyl's absorption maximum.

Evaluation of mixed Hg²⁺, Zn²⁺ and Cd²⁺ solutions

In order to determine the response of P1 to mixed solutions of Hg²⁺, Zn²⁺ and Cd²⁺, various ratios of the metals were added to P1 solutions. Initially, the enhancement and blue shift in dansyl's emission intensity for two metal solutions were monitored. Various ratios of Cd²⁺ : Zn²⁺ produced an enhancement factor and blue shift similar to what was observed for just Cd²⁺, which is expected since Cd²⁺ had a larger log K value. Various ratios of Hg²⁺ : Zn²⁺ and Hg²⁺ : Cd²⁺ produced an enhancement factor and blue shift similar to what was observed for Hg²⁺. Although Zn²⁺ and Cd²⁺ both individually had better FRET responses than Hg²⁺, Hg²⁺quenching of Trp may have the largest effect on the FRET response of P1. This same result was also observed when all three metals were simultaneously monitored.

Conclusions

A new fluorescent peptidyl chemosensor based on the mercury bindingprotein MerP with FRET capabilities was designed and quantitatively characterized. This study represents the first FRET-based fluorescence enhancement with the binding of Hg²⁺, a metal that is generally characterized by its propensity to quench fluorescence . As a general rule, the appearance of a fluorescence signal with a binding event will be inherently more sensitive than an approach based on quenching by an analyte due to shot noise. Unlike many previous examples of Hg²⁺ sensors, the peptidyl chemosensor functioned in aqueous solution at pH 7.0

Responses to Zn²⁺, Cd²⁺and Ag⁺ were also observed, with Zn²⁺ and Cd²⁺ binding producing the largest FRET enhancement response. No FRET response to the other transition, alkali and alkaline earth metals tested was observed. Although one might intuitively think that the largest response should come from Hg²⁺, the response is based on the change in proximity of the fluorophores as a result of metal binding and not on binding strength. Additionally, Hg²⁺-induced quenching of the Trpdonor as well as Hg²⁺ binding to amine -containing sites may be limiting the amount of FRET that can occur. A larger FRET enhancement for Hg²⁺ could potentially be obtained by using a different FRET pair. While two UV-excitable fluorophores were used in this study, other FRET pairs usable in the visible or infrared region could feasibly be implemented for in situ applications.

Acknowledgements

This work was supported, in part, by the Robert A. Welch Foundation and the Texas Hazardous Waste Research Center. We would like to thank Professor Jason Shear for use of the spectrofluorimeter and Klaus Linse, Sandra Smith and Michelle Gadush for their assistance in peptide synthesis.

References

1. L. Jarup, Br. Med. Bull., 2003, 68, 167–182.
2. L. Fabbrizzi and A. Poggi, Chem. Soc. Rev., 1995, 24, 197–202.
3. R. Kramer, Angew. Chem., Int. Ed., 1998, 37, 772–773.
4. R. E. Williams, P. J. Holt, N. C. Bruce and C. R. Lowe, in Biosensors for Environmental Monitoring, ed. U. Bilitewski and A. P. F. Turner, Harwood Academic Publishers, Amsterdam, 2000, pp. 213–225.
5. R. Corradini, A. Dossema, G. Galaverna, R. Marchelli, A. Panagia and G. Sartor, J. Org. Chem., 1997, 62, 6283–6289.
6. G. De Santis, L. Fabbrizzi, M. Licchelli, C. Mangano, D. Sacchi and N. Sardone, Inorg. Chim. Acta, 1997, 257, 69–76.
7. J. Yoon, N. E. Ohler, D. H. Vance, W. D. Aumiller and A. W. Czarnik, Tetrahedron Lett., 1997, 38, 3845–3848.
8. K. A. Mitchell, R. G. Brown, D. Yuan, S. C. Chang, R. E. Utecht and D. E. Lewis, J. Photochem. Photobiol., A, 1998, 115, 157–161.
9. L. Prodi, F. Bolletta, M. Montalti and N. Zaccheroni, Eur. J. Inorg. Chem., 1999, 455–460.
10. M. Beltramello, M. Gatos, F. Mancin, P. Tecilla and U. Tonellato, Tetrahedron Lett., 2001, 42, 9143–9146.
11. G. Klein, D. Kaufmann, S. Schurch and J.-L. Reymond, Chem. Commun., 2001, 561–562.
12. L. Prodi, M. Montalti, N. Zaccheroni, F. Dallavalle, G. Folesani, M. Lanfranchi, R. Corradini, S. Pagliari and R. Marchelli, Helv. Chim. Acta, 2001, 84, 690–706.
13. J. Bourson, F. Badaoui and B. Valeur, J. Fluoresc., 1994, 4, 275–277.
14. E. U. Akkaya and S. Turkyilmaz, Tetrahedron Lett., 1997, 38, 4513–4516.
15. J. Kawakami, H. Itoh, H. Mitsuhashi and S. Ito, Anal. Sci., 1999, 15, 617–618.
16. R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149–2154.
17. E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989, pp. 131–148.
18. N. L. Brown, Trends Biochem. Sci., 1985, 10, 400–403.
19. T. J. Foster, CRC Crit. Rev. Microbiol., 1987, 15, 117–140.
20. S. J. Opella, T. M. DeSilva and G. Veglia, Curr. Opin. Chem. Biol., 2002, 6, 217–223.
21. G. Veglia, F. Porcelli, T. DeSilva, A. Prantner and S. J. Opella, J. Am. Chem. Soc., 2000, 122, 2389–2390.
22. T. M. DeSilva, G. Veglia, F. Porcelli, A. Prantner and S. J. Opella, Biopolymers, 2002, 64, 189–197.
23. A. Torrado, G. K. Walkup and B. Imperiali, J. Am. Chem. Soc., 1998, 120, 609–610.
24. S. Bhattacharya and M. Thomas, Tetrahedron Lett., 2000, 41, 10313–10317.
25. A. Singh, L. Yao, W. C. Still and D. Sames, Tetrahedron Lett., 2000, 41, 9601–9605.
26. Y. Zheng, K. M. Gattas-Asfura, V. Konka and R. M. Leblanc, Chem. Commun., 2002, 2350–2351.
27. Y. Zheng, X. Cao, J. Orbulescu, V. Konka, F. M. Andreopoulos, S. M. Pham and R. M. Leblanc, Anal. Chem., 2003, 75, 1706–1712.
28. T. Mayr and T. Werner, Analyst, 2002, 127, 248–252.
29. P. Jiang and Z. Guo, Coord. Chem. Rev., 2004, 248, 205–229.
30. L. Fabbrizzi, M. Licchelli, P. Pallavicini, A. Perotti, A. Taglietti and D. Sacchi, Chem.–Eur. J., 1996, 2, 75–82.
31. L. Fabbrizzi, M. Licchelli, P. Pallavicini, A. Perotti and D. Sacchi, Angew. Chem., Int. Ed. Engl., 1994, 33, 1975–1977.
32. M. Y. Chae and A. W. Czarnik, J. Am. Chem. Soc., 1992, 114, 9704–9705.
33. D. A. Pearce, G. K. Walkup and B. Imperiali, Bioorg. Med. Chem. Lett., 1998, 8, 1963–1968.
34. H. A. Godwin and J. M. Berg, J. Am. Chem. Soc., 1996, 118, 6514–6515.
35. D. S. McClure, J. Chem. Phys., 1952, 20, 682–686.
36. G. Hennrich, H. Sonnenschein and U. Resch-Genger, J. Am. Chem. Soc., 1999, 121, 5073–5074.
37. K. Rurack, M. Kollmannsberger, U. Resch-Genger and J. Daub, J. Am. Chem. Soc., 2000, 122, 968–969.
38. K. Rurack, U. Resch-Genger, J. L. Bricks and M. Spieles, Chem. Commun., 2000, 2103–2104.
39. G. Hennrich, W. Walther, U. Resch-Genger and H. Sonnenschein, Inorg. Chem., 2001, 40, 641–644.
40. J. V. Mello and N. S. Finney, J. Am. Chem. Soc., 2005, 127, 10124–10125.
41. R. Martinez, A. Espinosa, A. Tarraga and P. Molina, Org. Lett., 2005, 7, 5869–5872.
42. H. Zhang, L. Han, K. A. Zachariasse and Y. Jiang, Org. Lett., 2005, 7, 4217–4220.
43. L. Prodi, C. Bargossi, M. Montalti, N. Zaccheroni, N. Su, J. S. Bradshaw, R. M. Izatt and P. B. Savage, J. Am. Chem. Soc., 2000, 122, 6769–6770.
44. X. Guo, X. Qian and L. Jia, J. Am. Chem. Soc., 2004, 126, 2272–2273.
45. A. Caballero, R. Martinez, V. Lloveras, I. Ratera, J. Vidal-Gancedo, K. Wurst, A. Tarraga, P. Molina and J. Veciana, J. Am. Chem. Soc., 2005, 127, 15666–15667.
46. G. Zhang, D. Zhang, S. Yin, X. Yang, Z. Shuai and D. Zhu, Chem. Commun., 2005, 2161–2163.
47. Y. Yang, K. Yook and J. Tae, J. Am. Chem. Soc., 2005, 127, 16760–16761.
48. Y. Zhao, Z. Lin, C. He, H. Wu and C. Duan, Inorg. Chem., 2006, 45, 10013–10015.
49. H. Zheng, Z. Qian, L. Xu, F. Yuan, L. Lan and J. Xu, Org. Lett., 2006, 8, 859–861.
50. S. H. Kim, J. S. Kim, S. M. Park and S. Chang, Org. Lett., 2006, 8, 371–374.
51. E. M. Nolan and S. J. Lippard, J. Am. Chem. Soc., 2003, 125, 14270–14271.
52. E. M. Nolan, M. E. Racine and S. J. Lippard, Inorg. Chem., 2006, 45, 2742–2749.
53. S.-K. Ko, Y.-J. Yang, J. Tae and I. Shin, J. Am. Chem. Soc., 2006, 128, 14150–14155.
54. S. Yoon, A. Albers, A. P. Wong and C. J. Chang, J. Am. Chem. Soc., 2005, 127, 16030–16031.
55. A. Ono and H. Togashi, Angew. Chem., Int. Ed., 2004, 43, 4300–4302.
56. B. R. White and J. A. Holcombe, Talanta, 2007, 71, 2015–2020.
57. T. Forster, Ann. Phys., 1948, 2, 55–75.
58. B. W. Van Der Meer, G. Coker, III and S. Y. S. Chen, Resonance Energy Transfer: Theory and Data, VCH Publishers, Inc., New York, 1994, p. 157.
59. B. M. Dunn, C. Pham, L. Raney, D. Abayasekara, W. Gillespie and A. Hsu, Biochemistry, 1981, 20, 7206–7211.
60. M. Gustiananda, J. R. Liggins, P. L. Cummins and J. E. Gready, Biophys. J., 2004, 86, 2467–2483.
61. J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1st edn, 1983, pp. 189–218.
62. US Environmental Protection Agency, Mercury Update: Impact on Fish Advisories, EPA Fact Sheet: EPA-823-F-01-011, EPA Office of Water, Washington, DC, 2001 (http://www.epa.gov/waterscience/fishadvice/mercupd.pdf).
63. A. B. Descalzo, R. Martinez-Manez, R. Radeglia, K. Rurack and J. Soto, J. Am. Chem. Soc., 2003, 125.
64. US EPA, http://www.epa.gov/safewater/contaminants/index.html#mcls, 2003.
65. S.-H. Hong and W. Maret, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 2255–2260.
66. F. Oton, A. Tarraga and P. Molina, Org. Lett., 2006, 8, 2107–2110.
67. G. Nucifora, L. Chu, T. K. Misra and S. Silver, Proc. Natl. Acad. Sci. U. S. A., 1989, 86, 3544–3548.
68. J. M. Berg, Acc. Chem. Res., 1995, 28, 14–19.
69. V. P. Culotta, L. W. J. Klomp, J. Strain, R. L. B. Casareno, B. Krems and J. D. Gitlin, J. Biol. Chem., 1997, 272, 23469–23472.
70. J.-M. Lehn and F. Montavon, Helv. Chim. Acta, 1978, 61, 67–82.
71. N. R. Cha, M. Y. Kim, Y. H. Kim, J.-I. Choe and S.-K. Chang, J. Chem. Soc., Perkin Trans. 2, 2002, 1193–1196.
72. R. F. Chen, Arch. Biochem. Biophys., 1971, 142, 552–564.

---