PEPTIDYL FLUORESCENT CHEMOSENSOR FOR DIVALENT ZINC

Abstract: The present invention provides a selective fluorescent chemosensor, sensitive to nanomolar concentrations of zinc (II) and selective for this ion over Na<+>, Mg<2+>, Co<2+>.

Description: TITLE OF THE INVENTION PEPTIDYL FLUORESCENT CHEMOSENSOR FOR DIVALENT ZINC The U.S. Government has certain rights in this invention pursuant to Grant No. CHE 9412442 awarded by the National Science Foundation. BACKGROUND OF THE INVENTION Field of the Invention The present invention provides a selective fluorescent chemosensor, sensitive to nanomolar concentrations of zinc(II), Zn 2 *, a device containing the same, and methods for measuring the concentration of a metal in a sample using the device. Discussion of the Backgrounds The selective and quantitative detection of trace amounts of divalent zinc (Zn 2 *) is commercially desirable fcr* ~ the diagnosis of metal ion induced disease states and in the protection of the environment. Zinc is an essential element which is present in the body at approximately 1 μmole/L. The USDA recommended dietary intake of Zn 2 * is only 15 mg/day, this provides an indication of how little Zn 2 * is required to maintain the required level of this element in a healthy adult. 1 Despite this relatively low concentration, Zn 2 * plays an essential role in biology and nutrition. Minor perturbations of normal Zn 2* levels have been associated with retarded sexual maturation, stunted growth and skin damage. Over 99% of Zn 2* in biological tissues and fluids is present in a chemically-combined form, with very little present as free Zn 2* ion. Traditional methods such as atomic absorption effectively measure total Zn 2* but can not distinguish between the chemically-combined and free forms. The problem of detecting free Zn 2 * is compounded because total free Zn 2 * is decreased only very slightly (50-100 pmol/10 6 cells) in cases of severe Zn 2 * deficiency. The detection of Zn 2 * in the environment is also important, and is presently an intractable problem. 2 For example, interest in Zn 2* concentrations in the ocean stems from its dual role as a required nanonutrient and as a potential toxic agent due to its widespread industrial and marine usage. Zinc exists at natural levels in ocean surface water at a total concentration of ca. 0.1 nM. 3 Dissolved Zn 2 * concentrations in seawater have been determined using atomic absorption spectrometry, mass spectrometry and voltammetry. The equipment for these procedures is shore based, time consuming and expensive. The concentration data are also inaccurate due to interference from other cations naturally present in sea water. A rapid, selective and more sensitive test for Zn 2 * concentrations which can be performed at sea is desirable. The current technologies for selectively detecting free Zn 2* in the presence of the chemically-combined forms have sensitivities in the micromolar range. For example, Zinquin 4 has a rather low sensitivity with association constants approximately 5 orders of magnitude weaker than many metal binding peptides and proteins. 5 In addition to its insensitivity, Zinquin cannot be used in the presence of copper. There remains a need in the art for probes which have excellent sensivity for Zn 2 * and high selectivity for this element in the presence of other transition metal cations. SUMMARY OF THE INVENTION Accordingly, one object of this invention is to provide a selective fluorescent chemosensor, sensitive to nanomolar concentrations of a metal cation. A second object of the present invention is to provide a device comprising the chemosensor and a detector. A third object of the present invention is to provide a method for detecting the presence of a metal in a sample using such a device. In light of the selectivity and avidity with which proteins bind Zn(II), 6 the present inventors used polypeptide architecture as the framework for metal ion recognition. 7 By manipulating synthetic polypeptides, the present inventors achieved the coordinated integration of fluorescent reporters for signal transduction within a metal binding peptidyl construct and obtained the chemosensor described herein. The present inventors have now developed a chemical probe which can provide direct, quantitative read-out of total free Zn 2 * concentrations in a fluid sample, either biological or seawater, similar to existing fiber optic sensors. 8 BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein: Figure 1. Schematic of the chemosensor design. Metal binding induces peptide folding, shielding a fluorophore from solvent and increasing emission intensity. Figure 2. Fluorescence emission response of ZNS1 to increasing levels of Zn 2* (μM) in the presence of Mg 2 * (50 mM) , and Co 2* (100 μM) ions; excitation at 333 nm. Spectra were acquired with 1.4 μM ZNS1, 50 mM HEPES, pH 7.0, μ=0.5 (NaCI), 3.5 μM EDTA. Addition of EDTA was required to complex adventitious metal and establish the initial baseline prior to metal addition. The relative increase in fluorescence at 475 nm is linear between 0.1 and 1.0 μM Zn 2 *. Figure 3. Ratiometric analysis of the fluorescence emission changes upon the addition of various divalent metals. Initial conditions are 1.4 μM ZNS1, 50 mM HEPES, pH 7.0, 3.5 μM EDTA, μ=0.5 (NaCI). Arrows represent the addition of concentrated metal stocks to give the indicated composition. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention comprises (i) a synthetic polypeptide template and (ii) a covalently attached fluorescent reporter that is sensitive to metal-induced conformational changes of the supporting framework, yet remote from the metal binding site. This approach combines the advantages of optical signalling 9 with the selective metal binding properties of an appropriate peptide. Furthermore, the modular design enables an approach wherein the fluorescent signalling mechanism is decoupled from the composition of the coordination sphere. As used herein, the term free Zn refers to Zn ions which are available. The term chemically-combined refers to Zn that is tightly bound, or coordinated, to a competing ligand. Metal Binding Peptide Templates Suitable metal binding peptide templates can be designed based on any naturally occurring metal binding protein. Preferred Zn binding peptidyl templates in accordance with the present invention are described in the following Table along with the protein they are components of and their SEQ ID NOs. Human SP1 QHICHI-QGCGKVYGKTSHLRAHLR—WHTG (SEQ ID NO:l) PFMCTW-SYCGKRFTRSDELQRHK —THTG (SEQ ID NO:2) KFACPE CPKRFMRSDHLSKHIK—THQN (SEQ ID NO:3) Drosophila Serendipity b EIPCHI CGEMFSSQEVLERHIKADTCQK (SEQ ID NO:4) QATCNV CGLKVKDDEVLDLHMN--LHEG (SEQ ID NO:5) ELECRY CDKKFSHKRNVLRHME— HWD (SEQ ID NO:6) KYQCDK CGERFSLSWLMYNHLM—RHDA (SEQ ID NO:7) ALICEV CHQQFKTKRTYLHHLR--THQT (SEQ ID NO:8) -YPCPD CEKSFVDKYTLKVHKR--VHQP (SEQ ID NO:9) Drosophila Serendipity g KQECTT CGKVYNSWYQLQKHLS-EEHSK (SEQ ID NO:10) NHICPI CGVIRRDEEYLELHMN—LHEG (SEQ ID NO:ll) EKQCRY CPKSFSRPVNTLRHM —SHWD (SEQ ID NO:12) KYQCEK CGLRFSQDNLLYNHR —RHEA (SEQ ID NO:13) PIICSI CNVSFKSRKTFNHHTL—IHKE (SEQ ID NO: 14) -HYCSV CPKSFTERYTLKMHMK—THEG (SEQ ID NO: 15) SGFCLI CNTTFENKKELEHHLQ--FHAD (SEQ ID NO: 16) Drosophila Kruppel SFTCKI CSRSFGYKHVLQNHER—THTG (SEQ ID NO: 17) PFECPE CDKRFTRDHHLKTHMR—LHTG (SEQ ID NO:18) PYHCSH CDRQFVQVANLRRHLR--VHTG (SEQ ID NO:19) PYTCEI CDGKFSDSNQLKSHML—^lTG (SEQ ID NO:20) PFECER CHMKFRRRHHLMNHK CGI (SEQ ID NO:21) Drosophila Snail RFKCDE CQKMYSTSMGLSKHRQ—FHCP (SEQ ID NO:22) THSCEE CGKLYTTIGALKMHIR HTL (SEQ ID NO:23) PCKCPI CGKAFSRPWLLQGHIR—THTG (SEQ ID NO:24) PFQCPD CPRSFADRSNLRAHQQ—THVD (SEQ ID NO:25) KYACQV CHKSFSRMSLLNKHSS-SNCTI (SEQ ID NO:26) Xenopus Xfin SHHCPH CKKSFVQRSDFLKHQR—THTG (SEQ ID NO:27) PYQCVE CQKKFTERSALVNHQR—THTG (SEQ ID NO:28) PYTCLD CQKTFNQRSALTKHR —THTG (SEQ ID NO:29) PYRCSV CSKSFIQNSDLVKHLR—THTG (SEQ ID NO:30) PYECPL CVKRFAESSALMKHKR—THST (SEQ ID NO:31) PFRCSE CSRSFTHNSDLTAHMR—KHTE (SEQ ID NO:32) PYSCSK CRKTFKRWKSFLNHQQ—THSR (SEQ ID NO:33) PYLCSH CNKGFIQNSDLVKHF —THTG (SEQ ID NO:34) PYQCAE CHKGFIQKSDLVKHLR--THTG (SEQ ID NO:35) PFKCSH CDKKFTERSALAKHQR— HTG (SEQ ID NO:36) PYKCSD CGKEFTQRSNLILHQR—IHTG (SEQ ID NO:37) PYKCTL CDRTFIQNSDLVKHQK—VHAN (SEQ ID NO:38) PHKCSK CDLTFSHWSTFMKHSK—LHSG (SEQ ID NO:39) KFQCAE CKKGFTQKSDLVKHIR—VHTG (SEQ ID NO:40) PFKCLL CKKSFSQNSDLHKHWR—IHTG (SEQ ID NO:41) PFFCYT CDKSFTERSALIKHHR—THTG (SEQ ID NO:42) PHKCSV CQKGFIQKSALTKHSR—THTG (SEQ ID NO:43) PYPCTQ CGKSFIQNSDLVKHQR—IHTG (SEQ ID NO:44) PYHCTE CNKRFTEGSSLVKHRR- -THSG (SEQ ID NO: 45) PYRCPQ CEKTFIQSSDLVKHLV- -VHNG (SEQ ID NO. 46) PYPCTE CGKVFHQRPALLKHLR- -THKT (SEQ ID NO 47) RYPCNE CSKEFFQTSDLVKHLR- -THTG (SEQ ID NO 48) PYHCPE CNKGFIQNSDLVKHQR- -THTG (SEQ ID NO 49) PYTCSQ CDKGFIQRSALTKHMR- -THTG (SEQ ID NO 50) PYKCEQ CQKCFIQNSDLVKHQR- -IHTG (SEQ ID NO 51) PYHCPD CDKRFTEGSSLIKHQR- -IHSR (SEQ ID NO 52) PYPCGV CGKSFSQSSNLLKHLK- -CHSE (SEQ ID NO 53) SFKCND CGKCFAHRSVLIKHVR- -IHTG (SEQ ID NO 54) PYKCGL CERSFVEKSALSRHQR- -VHKN (SEQ ID NO 55) RYSCSE CGKCFTHRSVFLKHWR- -MHTG (SEQ ID NO :56) PYTCKE CGKSFSQSSALVKHVR- -IHTG (SEQ ID NO 57) PYACST CGKSFIQKSDLAKHQR- -IHTG (SEQ ID NO :58) PYTCTV CGKKFIDRSSWKHSR- -THTG (SEQ ID NO .59) PYKCNE CTKGFVQKSDLVKHMR- -THTG (SEQ ID NO 60) PYGCNC CDRSFSTHSASVRHQR- -MCNT (SEQ ID NO :61) Drosophila Terminus DLHCRR CRTQFSRRSKLHIHQK-LRCGQ (SEQ ID NO:62) Yeast SW15 TFECLF-PGCTKTFKRRYNIRSHIQ—THLE (SEQ ID NO:63) PYSCDH-PGCDKAFVRNHDLIRHKK—SHQE (SEQ ID NO:64) -YACP CGKKFNREDALWHRSRMICSG (SEQ ID NO:65) Xenopus transcription factor IIIA RYICSF-ADCGAAYNKNWKLQAHLC—KHTG (SEQ ID NO:66) PFPCKE-EGCEKGFTSLHHTLRHSL—THTG (SEQ ID NO:67) NFTCDS-DGCDLRFTTKANMKKHFN-RFHNI (SEQ ID NO:68) VYVCHF-ENCGKAFKKHNQLKVHQF—SHTQ (SEQ ID NO:69) PYECPH-EGCDKRFSLPSRLKRHEK—VHAG (SEQ ID NO:70) -YPCKKDDSCSFVGKTWTLYLKHVA-ECHQD (SEQ ID NO:71) -AVCDV CNRKFRHKDYLRDHQK—THEK (SEQ ID NO:72) VYLCPR-DGCDRSYTTAFNLRSHIQ-SFHEE (SEQ ID NO:73) PFVCEH-AGCGKCFAMKKSLERHSV—VHDP (SEQ ID NO:74) Yeast ADR1 SFVCEV CTRAFARQEHLKRHY —SHTN (SEQ ID NO:75) PYPCGL CNRCFTRRDLLIRHAQ-KIHSG (SEQ ID NO:76) Drosophila KRH TYRCSE CQREFELLAGLKKHLK—THRT (SEQ ID NO:77) KYQCDI CGQKFVQKINLTHHAR—IHSS (SEQ ID NO:78) PYECPE CQKRFQERSHLQRHQK—YHAQ (SEQ ID NO:79) SYRCEK CGKMYKTERCLKVHNL—VHLE (SEQ ID NO:80) PFACTV CDKSFISNSKLKQHSN—IHTG (SEQ ID NO:81) PFKCNY CPRDFTNFPNWLKHTR-RRHKV (SEQ ID NO:82) Mouse MKR1 PFVCNY CDKTFSFKSLLVSHKR—IHTG (SEQ ID NO:83) PYECDV CQKTFSHKANLIKHQR—IHTG (SEQ ID NO:84) PFECPE CGKAFTHQSNLIVHQR—AHME (SEQ ID NO:85) PYGCSE CGKAFTHQSNLIVHQR—IHTG (SEQ ID NO:86) PYECNE CAKTRRKKSNLIIHQK—IHTG (SEQ ID NO:87) RYECNE CGKSFIQNSQLIIHR —THTG (SEQ ID NO:88) PYECTE CGKTFSQRSTLRLHLR—IHTG (SEQ ID NO:89) Mouse MKR2 VYGCDE CGKTFRQSSILLKHQR—IHTG (SEQ ID NO:90) PYTCNV CDKHFIERSSLTVHQR—THTG (SEQ ID N0:91) PYKCHE CGKAFSQSMNLTVHQR—THTG (SEQ ID N0:92) PYQCKE CGKAFRKNSSLIQHE —IHTG (SEQ ID NO:93) PYKCHD CEKAFSKNSSLTQHRR—IHTG (SEQ ID NO:94) PYECMI CGKHFTGRSSLTVHQV—IHTG (SEQ ID NO:95) PYECTE CGKAFSQSAYLIEHRR—IHTG (SEQ ID NO:96) PYECDQ CGKAFIKNSSLIVHQR—IHTG (SEQ ID NO:97) PYQCNE CGKPFSRSTNLTRHQR—THT- (SEQ ID NO:98) Drosophile Hunchback NYKCKT CGWAITKVDFWAHTR—THMK (SEQ ID NO:99) ILQCPK CPFVTERKHHLEYHIR—KHKN (SEQ ID NO:100) PFQCDK CSYTCVNKSMLNSHRK—SHSS (SEQ ID NO:101) QYRCAD CDYATKYCHSFKLHLRHYGHKP (SEQ ID NO:102) IYECKY CDIFFKDAVLYTIHMG—YHSC (SEQ ID NO:103) VFKCNM CGEKCDGPVGLFVHMARNAHS- (SEQ ID NO: 104) Trvpanosome TRS-1 PTKCTE CDATYQCRSSAVTHMV-NKHGF (SEQ ID NO:105) VLHCTI CASKFAVPGRLLHHLR-TIHGI (SEQ ID NO:106) PFQCDL CEASFGTHSSLSLHKK-LKHKS (SEQ ID NO:107) EVQCGV CQKVLSCRDSLIRHCK-AFHKG (SEQ ID NO: 108) MLV.— ■ 'T CGRQCASKTGLTLHQK-KMHGM (SEQ ID NO: 109) Mouse NGFI-A PYACPV-ESCDRRFSRSDELTRHIR—IHTG (SEQ ID NO:110) PFQCRI CMRNFSRSDHLTTHI —THTG (SEQ ID NO:111) PFACDI CGRFARSDERKKRHTK—IKLR (SEQ ID NO:112) Human ZFY VYPCMI CGKKFKSRGFLKRHMK—NHPE (SEQ ID NO:113) KYHCTD CDYTTNKKISLHNHLE—SHKL (SEQ ID NO:114) AIECDE CGKHFSHAGALFTHKM—VHKE (SEQ ID NO:115) MHKCKF CEYETAEQGLLNRHLL-AVHSK (SEQ ID NO:116) PHICVE CGKGFRHPSELRKHMR—IHTG (SEQ ID NO:117) PYQCQY CEYRSADSSNLKTHIK-TKHSK (SEQ ID NO:118) PFKCDI CLLTFSDTKEVQQHTL—VHQE (SEQ ID NO: 119) THQCLH CDHKSSNSSDLKRHVI-SVHTK (SEQ ID NO:120) PHDCEM CEKGFHRPSELKKHVA—VHKG (SEQ ID NO:121) MHQCRH CDFKIADPFVLSRHIL-SVHTK (SEQ ID NO: 122) PFRCKR CRKGFRQQNELKKHrøζ—THSG (SEQ ID NO:123) VYQCEY CEYSTTDASGFKRHVI-SIHTK (SEQ ID NO:124) PHRCEY CKKGFRRPSEKNQHIM—RHHK (SEQ ID NO:125) Xenopus P43 5S RNA binding protein LLRCPA-AGCKAFYRKEGKLQDHMA—GHSE (SEQ ID NO: 126) PWKCGI-KDCDKVFARKRQILKHVK—RHLA (SEQ ID NO:127) KLSCPT-AGCKMTFSTKKSLSRHKL-YKHGE (SEQ ID NO:128) PLKCFV-PGCKRSFRKKRALRRHLS—VHSN (SEQ ID NO:129) LSVCDV-PGCSWKSSSVAKLVAHQK—RHRG (SEQ ID NO:130) -YRCSY-EGCQTVSPTWTALQTHVK—KHPL (SEQ ID NO:131) -LQCAA CKKPFKKASALRRHKA—THAK (SEQ ID NO:132) QLPCPR-QDCDKTFSSVFNLTHHVARKLHLC (SEQ ID NO:133) THRCPH-SGCTRSFAMRESLLRHLV—VHDP (SEQ ID NO:134) All amino acid sequences shown in the above table and throughout the specification are represented by the standard one-letter code for amino acids. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine, W, tryptophan; Y, tyrosine. Dashes are present merely to allow alignment of the sequences. The above Zn peptidyl templates can be designed based on known metal binding domains in zinc-fingers. A database with the sequence for 135 such domains appears in "A Consensus Zinc Finger Peptide: Design, High-Affinity Metal Binding, a pH- Dependent Structure, and a His to Cys Sequence Variant," Krizek et al., J. Am. Chem. Soc. 1991, 113, 4518; incorporated herein by reference. A database with 1340 zinc finger domains appears in GH Jacobs, EMBO J. , 1992, 11, 4507-4517. The metal binding domain of the zinc finger proteins shown above and described throughout the literature can be represented by the consensus sequence: 1011 (R^-X-C-X^-C-Xj-R'-X s -R'-X.-H-Xj. s -H-X^g Where X is any α-amino acid (both natural and synthetic) and the subscript refers to the number of residues at each location. The residues R^R 3 shown in bold in the above consensus sequence are known to form a hydrophobic core which contributes to the proper folding of the domain. These residues are preferably maintained as hydrophobic residues for high affinity binding. Suitable hydrophobic residues include Phe, Tyr, Leu, His, He, Val, etc. Preferably, R 1 is Phe or Tyr; R 2 is Phe and R 3 is Leu. However, as mentioned above, these residues can also be replaced with any α-amino acid. It is well established that the R 1 , R 2 , R 3 and X residues can be any α-amino acid; for example, each residue was replaced with an Ala and the domain of a zinc finger protein still folded properly and bound zinc. 12 The peptidyl template can either consist of the whole domain shown above or a portion of those domains which bind Zn (herein referred to as the "Zn binding domain"). A Zn binding domain comprises at least 15 residues, preferably at least 20 residues, particularly preferably at least 25 residues, and may include the entire amino acid sequence of the Zn binding domain or, alternatively, a sequence comprised of at least those amino acid residues known to coordinate to Zn in their relative spatial array (the four residues in each sequence shown in bold text in the above Table) . That is, the Zn binding peptidyl template which is comprised of a portion of or the whole metal binding domain may contain amino acid substitutions within the natural sequence, so long as those substitutions do not totally destroy the Zn binding capacity of the domain. The selectivity and sensivity of the probe, that is its ability to detect free Zn versus chemically-combined Zn, can be conveniently varied by substituting non-coordinating amino acid residues. The metal coordinating amino acids can be replaced with other metal coordinating amino acids. This provides a means for conveniently tuning the selectivity and sensitivity of the probe. Suitable metal coordinating amino acids include natural amino acids s.uch as^cysteine, histidine, aspartic acid, and glutamic acid. Alternatively, unnatural amino acid residues which bind metals can be used. A variety of such residues have recently been described in the literature, including bipyridal-alanine, and phenanthrolyl-alanine. 13 The Zn binding template may also comprise, in addition to a portion of, or the whole metal binding domain, attachments on the N- and/or C- termini. For example, the metal binding domain may be extended with either additional amino acids or organic blocking groups. Any number of amino acids can be used to extend the termini so long as the extension does not totally destroy the Zn binding capacity of the Zn binding domain. Preferably, the extensions at the N- and/or C-termini inc ude the natural sequence of the metal binding protein surrounding the Zn binding domain. The primary sequences for each of the Zn binding domains in the above Table are known in the literature. The Zn binding template includes any fragment of the natural occurring protein which includes at least the Zn binding domain shown in the above Table. Suitable organic groups useful for extending the N- and C-terminus include organic blocking groups which are known in the art and include, for example, amines, carboxylic acids, etc. Alternatively, the organic group can be a polyfunctional compound useful for linking the metal binding domain to a support or the support itself. Suitable solid supports and polylinkers are well described as are the means for attaching these to peptides. 14 Suitable solid supports include, for example, silicon, gold, glass and quartz. Fluorescent Reporter Suitable fluorescent reporter groups include any fluorescent group with photophysical properties that are sensitive to environment, such as: The above residues can be synthesized as described in the art. 15 Fluorophores are known to be polarity dependent as described in the literature. 16 In the absence of divalent zinc, the zinc finger domains tend to be disordered. In contrast, when appropriate metal cations are present, coordination to the metal binding residues nucleates the formation of a defined tertiary structure including a cluster of conserved hydrophobic residues. To detect binding of a metal, the fluorophore is suitably placed within the sequence of the metal binding domain or at one of the ends. There is no limit to the distance between the fluorophore and the metal binding site provided that the metal binding event induces a significant change in the conformation of the polypeptide and causes the fluorophore to experience a new environment. The fluorophore can be incorporated into the peptidyl template using conventional peptide synthesis techniques (see for example, M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag: Berlin, 1984; Stewart & Young, Solid Phase Peptide Synthesis, Pierce: Rockford IL, 1984; incorporated herein by reference) . In general, residues containing a carboxylic acid, aryl chloride or a sulfonyl chloride group can be incorporated onto the N-terminus of the peptidyl template or, preferably, into any residue in the peptidyl template. For example, these fluorphores can be incorporated onto the side chain of a residue (for example, onto the amino group of a β-amino alanyl group) . As mentioned above, in the first embodiment of the invention, the present inventors examined Zn 2 * binding proteins. In the absence of metal ions, some Zn 2 * binding proteins are structurally disordered. 17 When Zn 2 * ions are present, coordination to cysteine and histidine residues nucleates the formation of a defined tertiary structure including a cluster of conserved hydrophobic residues (underlined in the above consensus sequence (I)). For example in the first embodiment of their invention, the present inventors replaced one of the hydrophobic residues with a microenvironment-sensitive fluorophore to allow the monitoring of the metal binding event. 18 The fluorophore used was chosen to allow the selective elaboration of a side chain amine at variable positions within a synthetic polypeptide. That is, a differentially protected 0-amino alanine (Baa) derivative, (S)-2,3-diamino-N α -9-fluorenylmethyl-oxycarbonyl- N β -allyloxycarbonyl propanoic acid (Fmoc-L-Baa(alloc)-OH) , was utilized. Compatibility of this residue with Fmoc solid phase peptide synthesis allows for orthogonal deprotection with a palladium catalyst. 19 The newly liberated amine is then available for fluorophore attachment, while leaving the remainder of the protected, peptide intact until the final resin deprotection. As a first embodiment, the zinc finger domain "ZFY- swap" 20 was chosen as a template, and Phe 10 of that sequence was chosen as the site for replacement with the dansylated Baa amino acid derivative. As described below, the peptide ZNS1 -16- Methods for detecting Zn(H) Methods for detecting the presence of Zn in a sample will depend on the nature of the chemosensor. If the chemosensor is in solution, the method comprises: contacting a sample with the chemosensor to form a test solution, irradiating the test solution at a wavelength sufficient to excite the fluorophore, measuring the fluorescence emission of the test solution, and determining the concentration of metal in said sample based on said fluorescence emission. The amount of Zn in the sample can be quantified by comparing the measured fluorescence against a standard sample with known Zn(II) concentration. Suitable samples include any aqueous solution including seawater, biological fluids such as plasma, blood, urine, etc. In this embodiment of the method, the means for irradiating the sample and the means for measuring the fluorescence emission of the test solution are ideally performed by the same means - preferably a fluorometer. A wide range of fluorometers are commercially available. If the chemosensor is immobilized onto a solid support as described above, the method of the present invention comprises: contacting the immobilized chemosensor with a sample, irradiating and measuring the fluorescent emission of said sample, and 15 NOT FURNISHED UPON FLLLNG determining the concentration of metal in said sample based on said fluorescent emission. Suitable samples are described above. In this embodiment, the device provides the means for irradiating the sample and measuring the Zn(II) concentration in the sample. In the first embodiment, the fluorescence emission properties of ZNSl were sensitive to the presence of nanomolar concentrations of Zn 2* (Figure 2) . The addition of Zn 2 * results in increased emission upon excitation of 333 nm, and the response (at 475 mn) is linear between 0.1 and 1 μM Zn 2 *. 23 The chemosensor was selective for Zn(II) by a factor of IO 7 over Na * , 10 6 over Mg 2* and 10 3 over Co 2 *. As one important application of the zinc sensor is for the analysis of sea water, preliminary studies were also carried out in the presence of metal cations including 0.5 M Na * , 50 mM Mg 2* and 100 μM Co 2 *. The chemosensor is compatible with these competing species. 24 Due to a blue-shift which occurs upon metal binding, dual-wavelength ratiometric analysis of the fluorescence emission using 525 nm and 650 nm readings were used for Zn 2 * quantitation in solution. 25 An example of the response obtained by this method and the minimal effect of competing ions is shown in Figure 3. The addition of EDTA to the complexed material demonstrates that the signalling mechanism of this peptide is readily reversible. The presence of two cysteine residues in ZNSl makes it incompatible with ions such as Cu 2 *, due to complications from dithiol oxidation. However, the modular nature of the design and the ready access to a variety of metal coordinating amino acids suggest strategies for remediating this limitation. The first embodiment of the chemosensor is described in detail below and demonstrates the feasibility of exploiting metal-dependent structure modulation as the basis for trace analyte detection. ZNSl shows excellent affinity for Zn 2 * relative to the chemical probe Zinquin. 26 1. W Kain and B Schwederski in Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life Wiley, 1994. 2. RB Thompson and ER Jones, Anal. Chem. 1993, 65:730-734. 3. JL Nowicki et al, Anal. Chem. 1994, 66:2732-2738. 4. PD Zalewski et al., Biochem. J. , 1993, 296:403-408. 5. PD Zalewski et al. , Chemistry and Biology, 1994, 1:153-161. 6. Fraα≥sto da Silva et al. , The Biological Chemistry of the Elements. Clarendon Press; New York, p. 561 (1993) . 7. For a protein-based sensor that uses a diffusible fluorophore to report divalent zinc see (a) Thompson et al., Anal. Biochem., vol. 227, pp. 123-128 (1995); and (b) Thompson, Anal. Chem., vol. 65, pp. 730-734 (1993). 8. RB Thompson, Circuits and Devices, 1994:14-21. 9. Czarnik in Fluorescent Chemosensors for Ion and Molecule Recognition, ACS, Washington, D.C, pp. 1-9 (1993). 10. Berg, Ace. Chem. Res., vol. 28, pp. 14-19 (1995); Klug et al., FASEB J. , vol. 9, pp. 597-604 (1995). 11. An alternate consensus sequence is observed for a subset of the zinc finger domains. M.A. Weiss et al, Biochemistry, Vθl.E29, pp. 5660-5664 (1990). 12. Michael et al., Proc. Natl. Acad. Sci. USA, 1992, 89: 4796- 4800. 13. Include, papers which describe the synthesis and incorporation of these amino acids - at all positions of substitution; incorporated herein by reference. 14. For a review see, MA Gallop et al., J. Med. Chem., 1994, 37:1233-1251. 15. For a review see, RP Haugland, "Covalent Fluorescent Probes", In Excited States of Biopolymers, RF Steiner, Ed., Plenum Press: NY, 1983; incorporated herein by reference. 16. JR Lakowicz, Effects of Solvents on Fluorescence Emission, In "Principles of Fluorescence Spectroscopy", Plenum Press, New York, 1983; pp 189. 17. Frankel et al., Proc. Natl. Acad. Sci. U.S.A., vol. 84, pp. 4841-4845 (1987); Eis et al., Biochemistry, vol. 32, p. 7981 (1993). 18. Lakowicz in Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 189-218 (1983). 19. Kates et al., Anal. Biochem., vol. 2/2, pp. 303-310 (1993). 20. Zinc finger domains with single residue replacements have been structurally characterized, showing the hydrophobic cluster is tolerant of substitution at position 10 (i.e., the second underlined residue in the consensus sequence) . Weiss et al., Biochemistry, vol. 29, pp. 9808-9813 (1990). 21. The peptide was purified by reversed phase (C 1B ) HPLC. The identity of ZNSl was confirmed by electrospray mass spec. 22. RB Thompson, Circuits & Devices, May 1994, 14-21. 23. CD and UV-vis metal titration experiments verify that ZNSl binds Co 2* with high affinity (K D 1-5 μM) . Competition analysis in the presence of 1.5 μM Zn 2* AND 100 μm Co 2 * reveals less than 5% change in the zinc-bound species, therefore the K D for Zn 2* is <1 nM. 24. Bruland in Trace Elements in Sea Water; J.P.ERilet et al; Academic Press, London, pp. 157-220 (1975) . 25. In the absence of metal ions the emission maximum is at 560nm. The Zn 2 * complex has an emission maximum at 525 nm. Since these maxima are not widely separated, ratiometric analysis is carried out at 525 and 650 nm. This analysis normalizes data against bias from sources such as varying instrument response. 26. Zalewski et al., Chem. Bio., vol. 1, pp. 153-161 (1994). Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. EXAMPLES Reagents used were purchased from Aldrich Chemical Co. or Fluka. Protected amino acid derivatives and reagents were obtained from Milligen (Perseptive) Biosearch, except FmocL- Baa(alloc)-OH which was synthesized as outlined below. Fluorescence experiments were performed on a SLM-Aminco SPF 500c spectrofluorometer at ambient temperature. UV-Vis spectra were obtained, on a §himadzu UV160U, or a Beckman DU 7500 recording spectrophotometer. All solutions and buffers employed in assays • ■ ere composed of high-purity (18 MΩ cm" 1 ) water obtained from a MILLI-Q (available from Millipore) deionization purification system, and were handled in acid-washed HDPE containers. To prevent metal catalyzed- and auto-oxidation of ZNS-1, buffer solutions were sparged with argon for one half hour then hydrogen for fifteen minutes immediately prior to use. Metal titrations were performed by the addition of aliquots of concentrated stock solutions. The concentration of these stocks were determined by complexometric titrations performed in triplicate with standard EDTA solutions (Aldrich) and an appropriate coloro etric indicator. 1 Synthβsis of Fmoc-L-Ba (alloc)-OH The amino acid derivative (4) was synthesized from the precursor N a -Boc-L-j8-amino alanine 2,3 via the route outlined in Scheme I. Scheme 1. Strategy for the synthesis of F oc-Baa(alloc)-OH. Boc-L-Baa (alloc) -OH (2) A solution of the Boc-a ine (l) was prepared by dissolving 4.33 g (21.2 mmol) in 100 mL of 50% v/v THF in deionized water, assisted by sonication at 50 °C. Sodium carbonate (2.46 g, 23.2 mmol) in 7 mL of water was added, and the resultant slurry was cooled to 0°C with constant stirring. Allyl chloroformate (2.25 L, 21.2 mmol) and diisopropyl-ethylamine (3.66 L, 21.2 mmol) were added in alternating portions over a period of 20 minutes, after which the ice bath was removed and the reaction vessel let stir an additional 1.5 hr. The organic solvent was removed under reduced pressure, and additional deionized water added to increase the volume to 250 L. Citric acid was added until pH 3.2, and the solution extracted with CH 2 C1 2 (5 x 200 mL) . The organic layers were combined, dried over MgS0 4 , and the solvent removed under reduced pressure to give a clear oil. Traces of contaminating lactam were separated by chromatography on silica gel (95:4:1, CHCl 3 /MeOH/AcOH) to afford 4.60 g (75% yield) of a clear, colorless oil. L-Baa (alloc) -OH ■ TFA (3) A 100-mL round bottom flask was charged with a stir bar, Boc-L-Baa(alloc)-OH (2, 4.55 g, 15.8 mmol) , and CH 2 C1 2 (15 ML) , then chilled to 0 °C with constant stirring. Trifluoroacetic acid (TFA, 10 mL, 130 mmol) was added drop wise over 15 minutes. After an additional 15 minutes the reaction was allowed to come up to room temperature and let stir for 2 hours. Excess TFA was removed under a stream of nitrogen, then the solvent was removed in vacuo to give a clear gum. Lyophilization of this residue from benzene gave 3 as a clear oil (4.63g, 97% yield). Fmoc-L-Baa (alloc) -OH (4) The carbamate amine 3 (370 mg, 1.21 mmol) was dissolved in 2:1 THF/H 2 0 (12 mL) , requiring the assistance of vigorous stirring and sonication. Diisopropylethylamine (DIEA, 465 μL, 2.66 mmol) was added until the solution was slightly basic to litmus. Under vigorous stirring, N-(9-Fluorenylmethoxy-carbonyl)succinimide (FmocONSu, 816 mg, 2.42 mmol) was added in alternating portions with DIEA (420 μL, 2.42 mmol) over 30 minutes. The reaction was allowed to stir an additional four hours. The organic phase was removed in vacuo, and the remaining aqueous solution was increased in volume to -100 L, and acidified with citric acid to pH 3. This was extracted with diet ' -yl ether (2 x 50 mL) , and the organic phase discarded. The aqueous phase was further extracted with CH 2 C1 2 (4 x 100 mL) , the organic phases combined, dried over anhydrous Na 2 S0 4 , and concentrated under reduced pressure to afford a clear oil. Purification by silica gel chromatography (95:5:3 CHCl 3 /MEOH/Ac0H) , followed by lyophilization from benzene gave 4 (465 mg, 96% yield) as a white powder. Peptide synthesis General . Peptides were synthesized on a Milligen 9050 peptide synthesizer at a 0.125 mmol scale. The support used was in all cases PAL-PEG-PS (Millipore) thus providing carboxyterminal primary amides. Couplings were performed at a concentration of 0.3 M acylating reagent and HOBT, in a volume sufficient to achieve a three-fold excess of amino acid to resin-bound acne. Pentafluorophenyl ester / 1- hydroxybenzotriazole (Pfp/HOBT) chemistry was employed for all residues except Fmoc-L-Baa(alloc)-OH which was coupled by in situ active ester generation using HOBT/N,N'-diisopropyl- carbodiimide (HOBT/DIPCDI) activation. All coupling reactions were allowed to proceed for greater than 45 minutes, and were monitored for completion with the Enhanced Monitoring Opfion (EMO, Milligen) . A coupling judged to be incomplete (< 97%) was double coupled for an additional 45 minutes. After successful coupling cycles, any residual amines were acylated by a 10 min wash with 0.3 M acetic anhydride/HOBT solution in 9:1 DMF/dichloromethane. Removal of the Fmoc group was performed with piperidine (20% v/v in DMF) with a standard wash duration of 7 minutes. This time was extended if an inefficient deblock was detected by EMO. After addition of the final residue, the amino-terminus was acetyl-capped (DMF/acetic anhydride/triethylamine, 4 mL:63 μL:94 μL) for 0.5 h then washed with DMF (5 x 10 L) and MEOH (5 x 10 m) . Residual solvent was removed under reduced pressure. Alloc removal. The method of Kates et al. 4 was employed with some minor modifications. A typical procedure for removal of the alloc group was as follows. Under a blanket of nitrogen, a 20-mL plastic, stoppered vial was charged with resin from the completed peptide synthesis (300 mg, 0.21 meq/g, 63 μmol) , and 5 mL of a solvent cocktail (CHC1 3 morpholine/AcOH, 90:5:5). The resin was allowed to swell 10 min, to which tetrakis(triphenyl-phosphine)palladium (200 mg, 173 μmol) was added under a blanket of nitrogen. The vial was capped, shielded from light, and placed on a wrist-action shaker at low speed for 2 h. The resin was filtered, washed with CHClj (5 x 10 mL) , and a palladiumchelating cocktail (DMF/diethyldithiocarbamic acid • 3H 2 0 / triethylamine, 25 mL / 225 mg / 250 μL) . Traces of this solution were removed with a basic wash (0.5% v/v triethylamine in DMF), and a final wash with methanol. The resin was transferred to a clean plastic vial, and the residual solvent removed under reduced pressure. Dansyl coupling. Lyophilized resin taken directly from the alloc-removal procedure (280 mg, 0.21 meq/g, 59 μmol), was allowed to swell in DMF (5 mL) . Dansyl chloride (159 mg, 590 μmol) was added, followed by triethylamine (82 L, 590 μmol) . The vial was capped under a blanket of nitrogen, and placed on a wrist-action shaker at low speed for 2 h. The resin was filtered, washed with a basic wash (5 x 10 mL, see above) , washed with DMF (5 x 10 mL) , then finally washed with MeOH (5 x 10 mL) . The resin was transferred to a clean 20 L polyethylene vial, and residual solvent was removed under reduced pressure prior to peptide cleavage. Peptide cleavage acid purification . Peptides were cleaved after fluorophore coupling using 10 mL of Reagent K 5 (trifluoroacetic acid / H 2 0 / ethanedithiol / thioanisole / phenol, 82.5/5/5/5/2.5) with a 2 h incubation period. The resin was filtered, concentrated to about 2 mL volume, and precipitated with ether/hexane 2:1 at -20°C for 30 min. The supernatant was decanted, and the peptides trituratsd with ether/hexane 2/1 (5 x 50 mL) . The resultant solid was resuspended in water (20 L) , lyophilized, and then purified by reverse phase (C lβ ) high performance liquid chromatography (HPLC), gradient elution with 0.1% v/v TFA in acetonitrile (solvent B) added to 0.1% v/v TFA in water (solvent A). The reduced (dithiol) and oxidized (intramolecular disulfide) forms of the peptide are separable by HPLC. Both forms of the peptide were collected and characterized, then combined and submitted together to a reduction protocol. Peptide reduction The reduction was performed by dissolving the combined HPLC purified peptides in 0.1 M phosphate buffer, pH 8.0, 50 MM dithiothreitol (DTT), lO M EDTA, 6 M guanidine, and incubating at 25 °C, 1 h. The reduction mixture was loaded onto a C 18 Sep-Pak (Millipore) , and buffer salts and DTT were eluted with 10 mL water. The reduced peptide was eluted with 30% acetonitrile in water. Acetonitrile was removed from this solution under a stream of nitrogen until the sample could be frozen at 0°C, whence it was lyophilized. This was resuspended in high-purity water that had been sparged with argon 0.5 h then hydrogen 0.5 h. to delay oxidation. The resulting stock solution was periodically analyzed by HPLC and no significant (> 2%) disulfide formation was detected. The concentration of stock solutions ZNS-1 was determined by reaction with Eliman's reagent, 2,5'-dithiobis(2- nitrobenzoic acid) (DTNB) 6 . These assays were performed in triplicate, with excellent agreement (< 5% error) between replicate runs. With this knowledge, the extinction coefficient of the dansyl chromophore was determined at 333 nm for ZNS-1 to be 5.3 x 10 3 M "1 cm" 1 in 50 mM Hepes, pH 7.0. This value was used for all subsequent quantitations of ZNS-1. Emission fluorescence assay. Assays were performed at pH 7.0 in 50 mM Hepes buffer, 0.5 M NaCI in a stoppered 750 μL (1 cm x 3mm) quartz fluorometer cell. The concentration of ZNS-1 in a given assay was determined by measurement of the unique absorbance of the dansyl chromophore (e 333 nm = 5.3 x 10 3 ) . Blank scans were taken against the same buffer immediately prior to ZNS-1 addition. Emission spectra were accumulated at I nm intervals with the following parameters: excitation wavelength = 333 nm, emission wavelength - 400-750 nm, excitation band pass 4 mm, emission band pass = 2 mm, lamp potential = 975 V, gain = 10, filter (time constant) 3. To mask the effects of adventitious metal ions present prior to the initiation of metal titration, small aliquots of EDTA were added (in increments of 0.5 μM) until overlaying emission spectra were observed, and the wavelength of maximum emission was 560 nm. The EDTA concentration at the initiation of the metal titration was never in excess of 4 μM (< 0.001% of the Mg 2* , and < 4% of the CO 2* subsequently added). With the concentrations of Zn 2* and CO 2* present over the course of a filtration, and the relative stability constants of EDTA for these species, at most 10% of the Zn 2* would be complexed by EDTA. 7 Divalent metal ions were added to the assay from stock solutions prepared in unbuffered water. These included a 2.83 M solution of MgCl 2 , a 0.0969 M solution of C0C1 2 , and a 100 PM solution of ZnCl 2 . References 1. "Vogel's Textbook of Quantitative Inorganic Analysis;" Bassett, J. ; Denney, R. C. ; Jeffery, G. H. Mendham, J.; William Clowes: London, 1978; pp 223-402. 2. Arnold et al. , J. Am. Chem. Soc. 1985, 107, 7105-9. 3. Kucharczyk et al. , Synth. Comm. 1989, 19, 1603-1609. 4. Kates et al. , Anal. Biochem. 1993, 212, 303-310. 5. King et al., Int. J. Pept. Protein Res. 1990, 36, 255- 266. 6. Riddles et al., Meth. Enzymol. 1983, 91, 49-60. 7. 0'Sullivan & Smithers, Meth. Enzymol. 1979, 63, 294. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims: WHAT IS CLAIMED AS NEW AND DESIRED TO BE SECURED BY LETTERS PATENT OF THE UNITED STATES IS: 1. A sensor for measuring the concentration of zinc(II) in a sample, comprising: (i) a peptide template comprised of a Zn(II) binding domain of a Zn(II) binding protein, wherein covalently attached to (ii) a fluorescent reporting group. 2. The sensor of Claim 1, wherein said Zn(ll) binding protein is a zinc finger protein. 3. The sensor of Claim 1, wherein said Zn(ll) binding domain is of the formula: R 1 -X-C-X a -C-X b -R 2 -X c -R 3 -X d -H-X e -H-X f wherein a is 2, 3 or 4; b is 1, 2 or 3; c is 1, 2, 3, 4 or 5; d is 1 or 2; e is 3, 4 or 5; f is 2, 3, 4, 5 or 6; and R 1 , R 2 , R 3 and each X are, independently, an α- amino acid. 4. The sensor of Claim 3, wherein R 1 , R 2 and R 3 are, independently, a hydrophobic α-amino acid. 5. The sensor of Claim 4, wherein R 1 , R 2 and R 3 are, independently, Phe, Tyr, Leu, lie, His or Val. 6. The sensor of Claim 3, wherein said zinc (II) binding domain has an amino acid sequence selected from SEQ ID N0:1- 135. 7. The sensor of Claim 6, wherein said zinc (II) binding domain has an amino acid sequence of SEQ ID NO:135. 8. The sensor of Claim 1, wherein said fluorescent reporter group is covalently attached to the N- or C-terminus of the peptidyl template. 9. The sensor of Claim 1, wherein said fluorescent reporter group is covalently attached to a side chain of an amino acid residue in the peptidyl template. 10. The sensor of Claim 3, wherein R 1 , R 2 or R 3 is an amino acid residue covalently attached to a fluorescent reporter group. 11. The sensor of Claim 10, wherein R 2 is 12. The sensor of Claim 7, wherein Phe 10 is replaced with a residue of the formula: 13 . A device comprising: the chemosensor of Claim 1, and a fluorescence detector. 14. A device comprising: the chemosensor of Claim 1 immobilized onto a solid support, a means for irradiating the chemosensor, and a means for detecting the fluorescence emission of said chemosensor. 15. The device of Claim 14, wherein said sensor is immobilized onto said solid support via the N-terminus of said peptidyl template. 16. The device of Claim 14, wherein said solid support is gold, quartz, silicon or glass. 17. The device of Claim 14, wherein said means for detecting fluoresence is a charged coupled device. 18. A method for measuring the concentration of Zn(II) in a sample, comprising the steps of: contacting the device of Claim 13 with a sample, irradiating the sample at a wavelength sufficient to excite the fluorophore, measuring the fluorescent emission of said sample, and determining the concentration of metal in said sample based on said fluorescence emission. 19. A method for measuring the concentration of ZN(II) in a sample, comprising the steps of: contacting the device of__Claim 14 with a sample, irradiating the sample at a wavelength sufficient to excite the fluorophore, measuring the fluorescence emission of said sample, and determining the concentration of metal in said sample based on said fluorescence emission.

Inventors: IMPERIALI BARBARA, WALKUP GRANT K, IMPERIALI, BARBARA, WALKUP, GRANT, K.
Applicants: CALIFORNIA INST OF TECHN, CALIFORNIA INSTITUTE OF TECHNOLOGY
Application year: 1997
Main classification: G01N21
IPC codes: G01N21