WO2000051721A2 - Combinatorial chelator array - Google Patents

Abstract

The present invention provides an array of chelating molecules, methods for preparing an array of chelating molecules, methods for selecting optimal chelating molecules using a combinatorial approach and methods for removing undesired ions from a solution.

Description

COMBINATORIAL CHELATOR ARRAY

FIELD OF THE INVENTION

The present invention is in the field of biological and chemical synthesis and processing. The present invention relates to arrays and methods for removing metals from a liquid sample.

The present application claims priority to U S. Provisional Application Serial No. 60/122,466 filed March 1, 1999.

BACKGROUND OF THE INVENTION

Organic chelates may be used for capturing radioactive species selectively from waste streams. Organic chelates provide a means to isolate otherwise difficult to control metal ions For example, Nakayami et al teach conjugating "Tc0₄ to an antibody using a multi-denate chelating agent (Nakayama et al , Bioconjugate Chemistry 10-9-17 (1999)) Penderson et al teach a completely peptide based "Tc chelator for use in tumor labeling (Penderson et al, J. Med. Chem. 39:1361-1371 (1996)) Dasaradhi et al teach using modified cahxarenes for the chelation of Th⁺⁴ (Dasaradhi et al , J Chem. Soc Perkin Trans. 2(6): 1 187-1 192 (1997)). Raymond et al teach extracting UO,^{^} from aqueous solution in to chloroform using a chelator (Raymond et al , Inorg. Chem. Acta 240(l-2):593-601 (1995)).

One difficulty associated with using chelators for removing radioactive waste is a problem of selectivity m remediation site environments. Many chelators bind a wide variety of metals and hence are unsuitable for selectively removing one dilute waste contaminant from a stream containing high concentrations of other interfering species Moreover, a myriad of different chemical environments are found at remediation sites. As a result, chelators must function in a wide range of diverse chemical environments. These diverse chemical environments make the development of any one generic chelator for a particular contaminant unlikely. For example, a chelator designed for the separation of strontium from waste containing calcium may work well in one waste stream only to fail m another due to variation in such factors as pH, ionic contamination (such as Na", K⁺, Ba⁺², Al⁺³), or organic impurities Thus, the design of a chelator for each specific contaminant in each specific chemical environment presents a new research problem. There is a need to provide a high-throughput means to prepare and to screen combinatorial libraries of organic chelators to determine selectivity and robustness to environmental variables.

There is considerable interest m producing selective metal chelators (Franczyk et al , J. Am. Chem. Soc 1 14(21 ): 8138-8146 ( 1992)). For example, the chelation of alkali metals by crown ethers and the construction of porphyπn complexes of many different metals is highly desirable However, there are still very few high selectivity chelating agents available, especially for the transura c ions

Designing a chelating agent with high affinity for selected metals presents an empirical problem akin to designing a small molecule inhibitor for an enzyme In the past decade, the search for enzyme inhibitors has been vastly accelerated by the use of combinatorial chemistry The field of combinatorial chemistry has enabled formation of very large libraries of drug candidate molecules and the HTS of these libraries for activity (Ehteshami et al , Chemical Abstracts 127: 1 13815) The development of a combinatorial library is often a time consuming and expensive endeavor It is an object of the present invention to minimize the time and effort required to determine a suitable chelator for particular ions in particular conditions It is a further object of the present invention to minimize the time and effort required to chelate ions m a sample

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods for making an array of one or more metal chelators. The methods of the present invention feature providing an array having at least one and preferably a plurality of ammo acids attached wherein one or more ammo acids has been modified to include a chelating or multidentate hgand. Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size m which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyπn or a mixture thereof.

In a second aspect, the present invention features an array having at least one and preferably a plurality of amino acids attached wherein one or more ammo acids has been modified to include a chelating or multidentate hgand. Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size m which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyπn or a mixture thereof In a third aspect, the present invention features methods for obtaining a chelating agent suitable for one or more particular ions in certain conditions. The methods of the present invention feature constructing a combinatorial library of chelator molecules. Such a combinatorial library may be produced on an array The array may then be screened to determine which chelators are suited for removing or extracting a particular metal from a specific environment. Those compounds that are found to be optimal metal chelators may then be placed on a diagnostic array This array of chelators may then be exposed to an environment from which metal ions are to be extracted. In a fourth aspect, the present invention provides a method for chelating one or more ions present in a sample comprising the step of placing a sample, preferably a liquid or gas sample, in contact with an array having at least one and preferably a plurality of ammo acids attached wherein one or more ammo acids has been modified to include a chelating or multidentate hgand Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size in which all hetero atoms consist of O, NH or S any combination, acetoacetamide, acetoacetic acid, a porphyrm or a mixture thereof

BRIEF DESCRIPTION OF THE FIGURES

FIGURES la and lb illustrate selective deprotection by electrochemically generated reagents (protons) generated at electrodes 1 and 4 to expose reactive functionalities (NH₂) on linker molecules (L) proximate electrodes 1 and 4 The substrate is shown m cross section and contains 5 electrodes.

FIGURES 2a and 2b illustrate the bonding of monomers (A) bearing protected chemical functional groups (P) with the deprotected linker molecules (bearing reactive functionalities) proximate electrodes 1 and 4.

FIGURES 3a and 3b illustrate selective deprotection by protons generated at electrodes 2 and 4 of a second set of reactive functionalities on the molecule and monomer proximate electrodes 2 and 4, respectively.

FIGURES 4a and 4b illustrate the bonding of monomers (B) bearing protected chemical functional groups (P) with the deprotected molecule and monomer proximate electrodes 2 and 4, respectively.

FIGURE 5 illustrates a 5 electrode substrate bearing all possible combinations of monomers (A) and (B). The linker molecule proximate electrode 1 has a protected dimer, e g , a dipeptide, containing two (A) monomers bonded thereto The linker molecule proximate electrode 2 has a protected dimer containing a (B) monomer bonded to the linker molecule (L) and a protected (A) monomer bonded to said (B) monomer. The linker molecule proximate electrode 3, which represents a control electrode, demonstrates a linker molecule where no synthesis occurs because no potential is applied to the proximate electrode. The linker molecule proximate electrode 4 has a protected dimer containing an (A) monomer bonded to a linker molecule (L) and a protected (B) monomer bonded to said (A) monomer. The linker molecule proximate electrode 5 has a protected dimer containing two (B) monomers bonded to a linker molecule (L).

FIGURE 6 depicts exemplary chelator molecules, compounds 1 -7 For instance, compound 1 could be used as a chelator molecule whereby it could be connected via attachment to one of the aromatic rings. Compound 4 represents a binaphthyl chelator, it could be attached either at one of the aromatic rings or via one of the R groups Compound 5 represents one of many polyhydroxylated species which could be attached for additional functionality Compound 6 represents an aceto acetate group An especially preferred R constituent could be the amine of the growing peptide or a carbon based attachment point to the system Compound 7 is similar having an attachment position in the middle providing a malonate derivative

FIGURE 7 shows how such an array according to the present invention is used to synthesize a pattern of molecules First, the chip is coated with a biocompatible porous membrane that allows molecules to flow freely between the bulk solvent and the electrode The chip is then immersed in a solution containing a precursor to the electrochemically-generated (ECG) reagent of interest For peptide synthesis, this is preferably an ECG-reagent to remove amino protecting groups A computer may then interface with the array to turn on the desired electrode pattern, and the precursor is electrochemically converted into the active species The electrochemically-generated (ECG) reagent, in turn, reacts with molecules immobilized to the membrane overlying the electrode

FIGURE 8 demonstrates a central feature of preferred arrays according to the present invention having the ability to confine the electrochemically generated (ECG) reagents to a region immediately adjacent to a selected microelectrode Here, a fluorescein dye has been immobilized covalently at individually addressed microelectrode locations The dye may be tightly confined to a checkerboard pattern and exhibits substantially no chemical crosstalk between active and inactive microelectrodes This level of localization of ECG reagents may be achieved by exploiting the physical chemistry of the solution in which the microelectrode array is immersed Such solutions usually contain buffers and scavengers that react with ECG reagents However, the rate at which ECG reagents are produced can overwhelm the ability of the solution to react with them m the small local area immediately proximate to the microelectrode As a result, chemistry that is mediated by ECG reagents occurs near selected microelectrodes, but there is no chemical crosstalk

FIGURE 9 demonstrates that once a suitable chelator has been found with tight and selective binding, it may be immobilized on a stable resm to create a material capable of removing selected contaminating metals from solution. Once the resm has absorbed its maximum load of the desired ion it may be removed and replaced with fresh resm

FIGURE 10 illustrates exemplary modifications to ammo acids where the ammo acids cysteine and serine are modified to include attached binding groups such as immodiacetic acid and diethylenetπamme that may be incorporated into a peptide-based chelating molecule

FIGURE 11 exemplifies an additional chelator found to be applicable for specific chemical environments FIGURE 12 demonstrates an exemplary array of chelating molecules according to the present invention

FIGURE 13 depicts a method of screening chip-based combinatorial chelator libraries according to the present invention. Here, a library on an array is exposed to a waste stream that is a remediation target Electrochemical HTS of the chelator library enables rapid identification of one or more appropriate chelator molecules for the targeted waste stream.

FIGURE 14 exemplifies the inclusion of a modified ammo acid At any point in the process, a modified amino acid containing a chelating functional group may be incorporated into the growing peptide sequence.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides methods for making an array of one or more metal chelators The methods of the present invention feature providing an array having at least one and preferably a plurality of ammo acids attached wherein one or more amino acids has been modified to include a chelating or multidentate hgand. Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size m which all hetero atoms consist of O, NH or S any combination, acetoacetamide, acetoacetic acid, a porphyπn or a mixture thereof Additional preferred chelator molecules are described m Figure 6. Those of skill in the art may choose any number of chelating agents well known in the art.

The present invention utilizes a combinatorial library of chelator molecules for developing selective ion chelation systems and ion detection arrays The present invention provides methods for producing an array of potential chelators useful for determining optimal chelators suited to removing or extracting one or more particular metals from a specific environment.

The present invention preferably utilizes a method for electrochemical placement of a material at a specific location on a substrate as described in United States Patent Application Serial Nos. 09/003,075 and 09/214,348, the disclosures of which are herein incorporated by reference, and international patent application numbers PCT/US97/1 1463 and PCT US99/00599, comprising the steps of providing a substrate having at its surface at least one electrode that is proximate to at least one molecule that is reactive with an electrochemically generated reagent, applying a potential to the electrode sufficient to generate electrochemical reagents capable of reacting to the at least one molecule proximate to the electrode, and producing a chemical reaction thereby. Such method allows production of an array of ammo acids wherein at least one ammo acid is modified to include at least one chelating ligands or moieties. In some preferred embodiments, the present invention utilizes a method for the electrochemical placement of a material at a specific location on a substrate comprising the steps of: providing a substrate having at its surface at least one electrode that is proximate to at least one molecule bearing at least one protected chemical functional group, applying a potential to the electrode sufficient to generate electrochemical reagents capable of deprotecting at least one of the protected chemical functional groups of the molecule, and bonding the deprotected chemical functional group with a monomer or a pre-formed molecule. According to the present invention, the monomer or molecule is normally an amino acid.

In other preferred embodiments, the present invention utilizes a method for electrochemical synthesis of an array of separately formed polymers on a substrate, which comprises the steps of: placing a buffering or scavenging solution in contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto, selectively deprotecting at least one protected chemical functional group on at least one of the molecules, bonding a first monomer having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule; selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group; bonding a second monomer having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule; and repeating the selective deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule and the subsequent bonding of an additional monomer to the deprotected chemical functional group until at least two separate polymers of desired length are formed on the substrate surface. According to the present invention, the monomer or molecule is normally an ammo acid and the polymer is a peptide

In additional preferred embodiments, the present invention utilizes a method for electrochemical synthesis of an array of separately formed polymers on a substrate, which comprises the steps of: placing a buffering or scavenging solution m contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto, selectively deprotecting at least one protected chemical functional group on at least one of the molecules; bonding a first monomer having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule; selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group; bonding a second monomer having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule: and repeating the selective deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule and the subsequent bonding of an additional monomer to the deprotected chemical functional group until at least two separate polymers of desired length are formed on the substrate surface According to the present invention, the monomer or molecule is normally an ammo acid and the polymer is a peptide.

In further preferred embodiments, the present invention utilizes a "getter" structure such as a second electrode proximate to the array of electrodes or proximate to each of the electrodes individually. Such a "getter" structure may reduce chemical crosstalk between adjacent electrodes and/or prolong the life of semiconductor circuitry. Various semiconductor circuitry may be placed m a manner to control electrodes individually or corporately according to any one of the methods that are well known in the art A "getter" structure in accordance with the present invention may be placed in an appropriate location either exposed to the external environment or internal to a semiconducting device.

By using the electrochemical techniques discussed herein, it is possible to place monomers, both those that can be used for polymer synthesis and those that can be decorated, and pre-formed molecules at small and precisely known locations on a substrate. It is therefore possible to synthesize polymers of a known chemical sequence at selected locations on a substrate. For example, m accordance with the presently disclosed invention, one can place ammo acids or nucleotides at selected locations on a substrate to synthesize desired sequences of amino acids or nucleotides in the form of, for example, peptides and ohgonucleotides, respectively.

In order to form the preferred peptide backbones of the present invention, monomer amino acids with or without linker molecules may be placed at a specific location on a substrate. The present invention features synthesizing amino acid polymers at a specific location on a substrate, and m particular polypeptides, by means of a solid phase polymerization technique, which generally involves the electrochemical removal of a protecting group from a molecule provided on a substrate that is proximate at least one electrode. An ammo acid, preferably a terminal amino acid, is modified to contain a chelator molecule or hgand thereby providing the ability to remove ions from a solution or environment.

Electrochemical reagents capable of electrochemically removing protecting groups from chemical functional groups on the molecule are generated at selected electrodes by applying a sufficient electrical potential to the selected electrodes Removal of a protecting group, or "deprotection," in accordance with the invention, occurs at selected molecules when a chemical reagent generated by the electrode acts to deprotect or remove, for example, an acid or base labile protecting group from the selected molecules. In one embodiment of the present invention, a terminal end of a monomer ammo acid, or linker molecule (. e , a molecule which "links," for example, a monomer to a substrate) is provided with at least one reactive functional group, which is protected with a protecting group removable by an electrochemically generated reagent The protecting group(s) is exposed to reagents electrochemically generated at the electrode and removed from the monomer, amino acid or linker molecule in a first selected region to expose a reactive functional group. The substrate is then contacted with a first monomer or pre-formed molecule, which bonds with the exposed functional group(s) This first monomer or pre-formed molecule may also bear at least one protected chemical functional group removable by an electrochemically generated reagent.

The monomers or pre-formed molecules can then be deprotected m the same manner to yield a second set of reactive chemical functional groups. A second monomer or pre-formed molecule, which may also bear at least one protecting group removable by an electrochemically generated reagent, is subsequently brought into contact with the substrate to bond with the second set of exposed functional groups Any unreacted functional groups can optionally be capped at any point during the synthesis process. The deprotection and bonding steps can be repeated sequentially at this site on the substrate until peptides or polymers of a desired sequence and length are obtained.

In another embodiment of the present invention, the substrate having one or more molecules bearing at least one protected chemical functional group bonded thereto is proximate an array of electrodes, which array is m contact with a buffering or scavenging solution. Following application of an electric potential to selected electrodes in the array sufficient to generate electrochemical reagents capable of deprotecting the protected chemical functional groups, molecules proximate the selected electrodes are deprotected to expose reactive functional groups, thereby preparing them for bonding. A monomer solution or a solution of pre-formed molecules, such as amino acids, peptides, proteins, etc. is then contacted with the substrate surface and the monomers or pre-formed molecules bond with the deprotected chemical functional groups.

Another sufficient potential is subsequently applied to select electrodes in the array to deprotect at least one chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group. A second monomer or pre-formed molecule having at least one protected chemical functional group is subsequently bonded to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule. The selective deprotection and bonding steps can be repeated sequentially until peptides or polymers of a desired sequence and length are obtained. The selective deprotection step is repeated by applying another potential sufficient to effect deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule The subsequent bonding of an additional monomer or pre-formed molecule to the deprotected chemical functional group(s) until at least two separate polymers of desired length are formed on the substrate FIGURES 1-5 geneπcally illustrate the above-discussed embodiments.

Preferred embodiments of the methods of the present invention use a buffering or scavenging solution in contact with each electrode, which is buffered towards the electrochemically generated reagents, in particular, towards protons and/or hydroxyl ions, and that actively prevents chemical cross-talk caused by diffusion of the electrochemically generated ions from one electrode to another electrode in an array For example, when an electrode exposed to an aqueous or partially aqueous media is biased to a sufficiently positive (or negative) potential, protons (or hydroxyl ions) are produced as products of water hydrolysis Protons, for example, are useful for removing electrochemical protecting groups from several molecules useful m combinatorial synthesis, for example, peptides

In order to produce separate and pure polymers, it is desirable to keep these protons (or hydroxyl ions) confined to the area immediately proximate the selected electrode(s) m order to minimize, and, if possible to eliminate, chemical cross-talk between nearby electrodes in an array. The spatial extent of excursion of electrochemically generated reagents can be actively controlled by the use of a buffering or scavenging solution that reacts with the reagents that move away from the selected electrodes, thus preventing these reagents from reacting at a nearby electrode.

Another technique for confining these electrochemically generated reagents to the area immediately proximate the selected electrode(s) is to place a "getter" structure in proximity to the selected electrode(s) and substantially exposed to the external environment Such a "getter" structure may be used in conjunction with or in place of a scavenging solution A "getter" structure may be designed of any suitable material and formed into any suitable shape or size as skilled artisans will readily appreciate. The most important criteria for such a "getter" structure is that it function to scavenge electrochemically generated reagents that may diffuse away from the selected electrode(s). The "getter" structure may function passively by reacting chemically with the electrochemically generated reagents Alternatively, the "getter" structure may function actively to scavenge the electrochemically generated reagents This may be performed by applying sufficient potential to the "getter" structure to cause electrochemical scavenging Another function of the "getter" structure may be to prevent the diffusion of ions toward or into circuitry such as transistors that may be operably linked to the selected electrode(s). In accordance with this function, the "getter" structure may be placed substantially at the interface between an insulating dielectric and a metallization layer operably linked to the selected electrode(s). The substrate in the .m ention is proximate to at least one electrode, i e an electrically conducting region of the substrate that is substantially surrounded by an electrically insulating region The electrode(s), by being "proximate" to the substrate, can be located at the substrate, i e , embedded in or on the substrate, can be next to, below, or above the substrate, but need to be in close enough proximity to the substrate so that the reagents electrochemically generated at the electrode(s) can accomplish the desired deprotection of the chemical functional groups on the monomer(s) and/or molecule(s)

In addition to being proximate to at least one electrode, the substrate has on a surface thereof, at least one molecule, and preferably several molecules, bearing at least one chemical functional group protected by an electrochemically removable protecting group These molecules bearing protected chemical functional groups also need to be proximate to the electrode(s) In this regard, the molecules on the surface of the substrate need to be m close enough proximity to the electrode(s) so that the electrochemical reagents generated at the electrode can remove the protecting group from at least one protected functional group on the proximate molecule(s)

The molecules bearing a protected chemical functional group that are attached to the surface of the substrate may be selected generally from monomers, linker molecules and pre-formed molecules Preferably, the molecules attached to the surface of the substrate include monomers, ammo acids, peptides, and linker molecules All of these molecules generally bond to the substrate by covalent bonds or ionic interactions Alternatively, all of these molecules can be bonded, also by covalent bonds or ionic interactions, to a layer overlaying the substrate, for example, a permeable membrane layer, which layer can be adhered to the substrate surface in several different ways, including covalent bonding, ionic interactions, dispersive interactions and hydrophilic or hydrophobic interactions In still another manner of attachment, a monomer or pre- formed molecule may be bonded to a linker molecule that is bonded to either the substrate or a layer overlaying the substrate

The monomers, linker molecules and pre-formed molecules used herein, are preferably provided with a chemical functional group that is protected by a protecting group removable by electrochemically generated reagents If a chemical functional group capable of being deprotected by an electrochemically generated reagent is not present on the molecule on the substrate surface, bonding of subsequent monomers or pre-formed molecules cannot occur at this molecule Preferably, the protecting group is on the distal or terminal end of the linker molecule, monomer, or pre-formed molecule, opposite the substrate That is, the linker molecule preferably terminates in a chemical functional group, such as an amino or carboxy acid group, bearing an electrochemically removable protective group Chemical functional groups that are found on the monomers, linker molecules and pre-formed molecules include any chemically reactive functionality Usually, chemical functional groups are associated with corresponding protective groups and will be chosen or utilized based on the product being synthesized The molecules of the invention bond to deprotected chemical functional groups by covalent bonds or ionic interactions.

Monomers, particularly amino acids, used in accordance with the methods of the present invention to synthesize the various polymers, particularly peptides, contemplated include all members of the set of small molecules that can be joined together to form a polymer. This set includes, but is not limited to, the set of common L-ammo acids, the set of D-ammo acids and the set of synthetic amino acids Monomers include any member of a basis set for synthesis of a polymer. For example, tπmers of L-ammo acids form a basis set of approximately 8000 monomers for synthesis of polypeptides Different basis sets of monomers may be used at successive steps in the synthesis of a polymer The number of monomers that can be used in accordance with the synthesis methods can vary widely, for example from 2 to several thousand monomers can be used, but in more preferred embodiments, the number of monomers will range from approximately 4 to approximately 200, and, more preferably, the number of monomers will range from 4-20.

In a preferred embodiment of the invention, the monomers are ammo acids containing a protective group at its amino or carboxy terminus that is removable by an electrochemically generated reagent. A polymer m which the monomers are alpha ammo acids and are joined together through amide bonds is a peptide, also known as a polypeptide In the context of the present invention, it should be appreciated that the amino acids may be the L-optical isomer or the D-optical isomer or a mixture of the two. Peptides are at least two ammo acid monomers long, and often are more than 20 amino acid monomers long.

Furthermore, essentially any pre-formed molecule can be bonded to the substrate, a layer overlaying the substrate, a monomer or a linker molecule. Pre- formed molecules include, for example, proteins, including in particular, receptors, enzymes, ion channels, and antibodies, nucleic acids, polysacchaπdes, porphyπns, and the like Pre-formed molecules are, in general, formed at a site other than on the substrate of the invention. In a preferred embodiment, a pre-formed molecule is bonded to a deprotected functional group on a molecule, monomer, or another pre- formed molecule. In this regard, a pre-formed molecule that is already attached to the substrate may additionally bear at least one protected chemical functional group to which a monomer or other pre-formed molecule may bond, following deprotection of the chemical functional group.

Protective groups are materials that bind to a monomer, a linker molecule or a pre-formed molecule to protect a reactive functionality on the monomer, linker molecule or pre-formed molecule, which may be removed upon selective exposure to an activator, such as an electrochemically generated reagent Protective groups that may be used in accordance with the present invention preferably include all acid and base labile protecting groups. For example, peptide amine groups are preferably protected by t-butyloxycarbonyl (BOC) or benzyloxycarbonyl (CBZ), both of which are acid labile, or by 9-fluorenylmethoxycarbonyl (FMOC), which is base labile. Additionally, hydroxy groups on phosphoramidites may be protected by dimethoxytπtyl (DMT), which is acid labile Exocychc amine groups on nucleosides, in particular on phosphoramidites, are preferably protected by dimethylformamidine on the adenosine and guanos e bases, and isobutyryl on the cytidme bases, both of which are base labile protecting groups. This protection strategy is known as fast ohgonucleotide deprotection (FOD) Phosphoramidites protected m this manner are known as FOD phosphoramidites.

Additional protecting groups that may be used in accordance with the present invention include acid labile groups for protecting amino moieties: tert-butyloxycarbonyl, tert-amyloxycarbonyl, adamantyloxycarbonyl, 1 -methylcyclobutyloxycarbonyl, 2-(p-bιphenyl)propyl(2)oxycarbonyl, 2- (p-phenylazophenylyl)propyl(2)oxycarbonyl, α,α-dιmethyl- 3,5-dιmethyloxybenzyloxy-carbonyl, 2-phenylpropyl(2)oxycarbonyl, 4- methyloxybenzyloxycarbonyl, benzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl (trityl), p-toluenesulfenylaminocarbonyl, dimethylphosphmothioyl, diphenylphosphmothioyl, 2-benzoyl-l-methylvιnyl, o-mtrophenylsulfenyl, and 1- naphthyhdene; as base labile groups for protecting ammo moieties: 9- fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and 5- benzisoazolylmethyleneoxycarbonyl; as groups for protecting amino moieties that are labile when reduced: dithiasuccinoyl, p-toluene sulfonyl, and pipeπdmo-oxycarbonyl; as groups for protecting amino moieties that are labile when oxidized, (ethylthιo)carbonyl; as groups for protecting ammo moieties that are labile to miscellaneous reagents, the appropriate agent is listed parenthesis after the group: phthaloyl (hydrazme), tπfluoroacetyl (pipeπdme), and chloroacetyl (2- ammothiophenol); acid labile groups for protecting carboxyhc acids: tert-butyl ester; acid labile groups for protecting hydroxyl groups: dimethyltπtyl; and basic labile groups for protecting phosphotπester groups- cyanoethyl.

As mentioned above, any unreacted deprotected chemical functional groups may be capped at any point duπng a synthesis reaction to avoid or to prevent further bonding at such molecule. Capping groups "cap" deprotected functional groups by, for example, binding with the unreacted amino functions to form amides Capping agents suitable for use in the present invention include: acetic anhydride, n-acetyhmidizole, isopropenyl formate, fluorescamine , 3-nιtrophthalιc anhydride and 3-sulfopropomc anhydride. Of these, acetic anhydride and n-acetyhmidizole are preferred. The molecules of the invention, . e the monomers, linker molecules and pre- formed molecules, can be attached directly to the substrate or can be attached to a layer or membrane of separating material that overlays the substrate Materials that can form a layer or membrane overlaying the substrate, such that molecules can be bound there for modification by electrochemically generated reagents, include controlled porosity glass (CPG), generic polymers, such as, teflons, nylons, polycarbonates, polystyrenes, polyacylates, polycyanoacrylates, polyvinyl alcohols, polyamides, polyimides polysiloxanes, polysihcones, polymtriles, polyelectrolytes, hydrogels, epoxy polymers' melammes, urethanes and copolymers and mixtures of these and other polymers, biologically derived polymers, such as, polysacchaπdes, polyhyaluπc acids, celluloses, and chitons, ceramics, such as, alumina, metal oxides, clays, and zeolites, surfactants, thiols, self-assembled monolayers, porous carbon, and fullerine materials The membrane can be coated onto the substrate by spin coating, dip coating or manual application, or any other art acceptable form of coating

Reagents that can be generated electrochemically at the electrodes fall into two broad classes oxidants and reductants There are also miscellaneous reagents that are useful in accordance with the invention Oxidants that can be generated electrochemically include iodine, lodate, periodic acid, hydrogen peroxide, hypochlorite, metavanadate, bromate, dichromate, cerium (IV), and permanganate Reductants that can be generated electrochemically include chromium (II), ferrocyamde, thiols, thiosulfate, titanium (III), arsenic (III) and iron (II) The miscellaneous reagents include bromine, chloride, protons and hydroxyl ions Among the foregoing reagents, protons, hydroxyl ions, iodine, bromine, chlorine and the thiols are preferred

In accordance with preferred embodiments of the synthesis methods of the present invention, a buffering and/or scavenging solution is in contact with each electrode The buffering and/or scavenging solutions that may be used m accordance with the invention are preferably buffered toward, or scavenge, ions such as protons and/or hydroxyl ions, although other electrochemically generated reagents capable of being buffered and/or scavenged are clearly contemplated The buffering solution functions to prevent chemical cross- talk due to diffusion of electrochemically generated reagents from one electrode an array to another electrode in the array, while a scavenging solution functions to seek out and neutralize/deactivate the electrochemically generated reagents by binding or reacting with them Thus, the spatial extent of excursion of electrochemically generated reagents can be actively controlled by the use of a buffering solution and/or a scavenging solution In accordance with the invention, the buffering and scavenging solutions may be used independently or together Preferably, a buffering solution is used because the capacity of a buffering solution is more easily maintained, as compared with a scavenging solution Buffering solutions that can be used in accordance w ith the present invention include all electrolyte salts used m aqueous or partially aqueous preparations Buffering solutions preferably used in accordance w ith the present invention include acetate buffers, which typically buffer around pH 5, borate buffers, which typically buffer around pH 8, carbonate buffers, which typically buffer around pH 9, citrate buffers, which typically buffer around pH 6, glycme buffers, which typically buffer around pH 3, HEPES buffers, which typically buffer around pH 7, MOPS buffers, which typically buffer around pH 7. phosphate buffers, which typically buffer around pH 7, TRIS buffers, which typically buffer around pH 8, and 0 1 M KI in solution, which buffers the iodine concentration by the equilibrium reaction I₂ + I = I₃ , the equilibrium coefficient for this reaction being around 10 '

Alternatively, or in combination with a buffering solution, a sca\ enging solution may be used that contains species such as ternary amines that function as proton scavengers or sulfonic acids that function as hydroxyl ion scavengers in nonaqueous media The rate at which a reagent/species is scavenged depends both on the intrinsic rate of the reaction occurring and on the concentration of the scavenger For example, solvents make good sca\ engers because they are frequently present m high concentrations Most molecules scavenge in a nonselective way, however, some molecules, such as superoxide dismutase and horseradish peroxidase, scavenge in a selective manner

Of particular interest to the present invention are scavenger molecules that can scavenge the different reactive species commonly generated, for example, by water hydrolysis at electrodes, including hydroxyl radicals, superoxides, oxygen radicals, and hydrogen peroxide Hydroxyl radicals are among the most reactive molecules known, their rate of reaction is diffusion controlled, that is, they react with the first reactant/species they encounter When hydroxyl radicals are generated by water hydrolysis, the first molecule they usually encounter is a water molecule For this reason, water is a rapid and effective scavenger of hydroxyl radicals Superoxides are also a relatively reactive species, but can be stable m some nonaqueous or partially aqueous solvents In aqueous media, superoxides rapidly react with most molecules, including water In many solvents, they can be scavenged selectively with superoxidase dismutase

Oxygen radicals are a family of oxygen species that exist as free radicals They can be scavenged by a wide variety of molecules such as water or ascorbic acid Hydrogen peroxide is a relatively mild reactive species that is useful, m particular, in combinatorial synthesis Hydrogen peroxide is scavenged by water and many types of oxidizing and reducing agents The rate at which hydrogen peroxide is scavenged depends on the redox potential of the scavenger molecules being used Hydrogen peroxide can also be scavenged selectively by horseradish peroxidase Another electrochemically generated species that can be scavenged is iodine Iodine is a mild oxidizing reagent that is also useful for combinatorial synthesis Iodine can be scavenged by reaction with hydroxyl ions to form iodide ions and hypoiodite. The rate at which iodine is scavenged is pH dependent, higher pH solutions scavenge iodine faster. All of the scavenger molecules discussed above may be used in accordance with the present invention Other scavenger molecules will be readily apparent to those skilled m the art upon review of this disclosure.

In accordance with the synthesis methods of the present invention, the buffering solutions are preferably used in a concentration of at least 0 01 mM. More preferably, the buffering solution is present in a concentration ranging from 1 to lOOmM. and still more preferably, the buffering solution is present in a concentration ranging from 10 to lOOmM. Most preferably, the buffering solution concentration is approximately 30 mM. A buffering solution concentration of approximately 0 1 molar, will allow protons or hydroxyl ions to move approximately 100 angstroms before buffering the pH to the bulk values Lower buffering solution concentrations, such as 0.00001 molar, will allow ion excursion of approximately several microns, which still may be acceptable distance depending on the distance between electrodes in an array.

In accordance with the methods of the present invention, the concentration of scavenger molecules a solution will depend on the specific scavenger molecules used since different scavenging molecules react at different rates. The more reactive the scavenger, the lower the concentration of scavenging solution needed, and vice versa. Those skilled in the art will be able to determine the appropriate concentration of scavenging solution depending upon the specific scavenger selected.

The at least one electrode proximate the substrate of the invention is preferably an array of electrodes. Arrays of electrodes of any dimension may be used, including arrays containing up to several million electrodes. Preferably, multiple electrodes in an array are simultaneously addressable and controllable by an electrical source. More preferably, each electrode is individually addressable and controllable by its own electrical source, thereby affording selective application of different potentials to select electrodes in the array. In this regard, the electrodes can be described as "switchable".

The methods described herein are particularly suited to synthesizing a peptide having one or more modified amino acids, preferably a modified terminal amino acid, having one or more chelating ligands or moieties. Normally, such synthesis is accomplished by providing a modified amino acid as a monomer for including in a peptide chain Thus, the synthesis methods of the present invention allow the rapid and systematic synthesis of arrays of chelating agents attached to peptide backbones on an array. In a second aspect, the present invention features an array having at least one and preferably a plurality of amino acids attached wherein one or more amino acids is modified to include a chelating or multidentate hgand Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size in which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyrm or a mixture thereof. Additional preferred chelator molecules are described in Figure 6. Those of skill m the art may choose any number of chelating agents well known in the art.

The arrays of the present invention are preferably similar to those described in United States Serial Nos. 09/003,075 and 09/214,348, the disclosures of which are herein incorporated by reference. Such arrays allow synthesizing chemical compounds at well-defined and individually addressable locations. Such arrays may be manufactured at low cost by contract fabricators using existing semiconductor manufacturing facilities. Figure 7 describes how such a chip may be used to synthesize a pattern of molecules. First, the array may be coated with a biocompatible porous membrane that allows molecules to flow freely between a bulk solvent and the electrode. The array may then be immersed a solution containing a precursor to the electrochemically-generated (ECG) reagent of interest. For peptide synthesis, this is preferably an ECG-reagent to remove amino protecting groups. A computer may then interface with the array to turn on the desired electrode pattern, and the precursor is electrochemically converted into the active species. The electrochemically-generated (ECG) reagent, in turn, reacts with molecules immobilized to the membrane overlying the electrode.

A central feature of preferred arrays according to the present invention is the ability to confine the ECG reagents to a region immediately adjacent to a selected microelectrode. This is illustrated in Figure 8. Here, a fluorescein dye has been immobilized covalently at individually addressed microelectrode locations The dye may be tightly confined to a checkerboard pattern and exhibits substantially no chemical crosstalk between active and inactive microelectrodes. This level of localization of ECG reagents may be achieved by exploiting the physical chemistry of the solution in which the microelectrode array is immersed. Such solutions usually contain buffers and scavengers that react with ECG reagents. However, the rate at which ECG reagents are produced can overwhelm the ability of the solution to react with them the small local area immediately proximate to the microelectrode. As a result, chemistry that is mediated by ECG reagents occurs near selected microelectrodes, but there is no chemical crosstalk

In some embodiments, the surface of the array may be provided with a layer of linker molecules. Linker molecules allow for indirect attachment of monomers or pre-formed molecules to the substrate or a layer overlaying the substrate. The linker molecules are preferably attached to an overlaying layer via silicon-carbon bonds, using, for example, controlled porosity glass (CPG) as the layer material. Linker molecules also facilitate target recognition of the synthesized polymers Furthermore, the linker molecules are preferably chosen based upon their hydrophihc/hydrophobic properties to improve presentation of synthesized polymers to certain receptors For example, in the case of a hydrophilic receptor, hydrophilic linker molecules will be preferred so as to permit the receptor to approach more closely the synthesized polymer.

The linker molecules are preferably of sufficient length to permit polymers on a completed substrate to interact freely with binding entities exposed to the substrate The linker molecules, when used, are preferably 10 to 1000 atoms long, and in especially preferred embodiments are about 650 atoms long to provide sufficient exposure of the functional groups to the binding entity. The linker molecules, which may be advantageously used m accordance with the invention include, for example, aryl acetylene, ethylene glycol ohgomers containing from 2 to 10 monomer units, diammes, diacids, ammo acids, and combinations thereof. Other linker molecules may be used m accordance with the different embodiments of the present invention and will be recognized by those skilled the art in light of this disclosure.

According to another preferred embodiment, linker molecules may be provided with a cleavable group at an intermediate position, which group can be cleaved with an electrochemically generated reagent. This group is preferably cleaved with a reagent different from the reagent(s) used to remove the protective groups. This enables removal of the various synthesized polymers or nucleic acid sequences following completion of the synthesis by include: acetic anhydride, n-acetyhmidizole, isopropenyl formate, fluorescamine , 3-nιtrophthalιc anhydride and 3-sulfoproponιc anhydride. Of these, acetic anhydride and n-acetyhmidizole are preferred.

The linker molecules are preferably of sufficient length to permit polymers on a completed substrate to interact freely with binding entities exposed to the substrate. The linker molecules, when used, are preferably 650 atoms long to provide sufficient exposure of the functional groups to the binding entity. The linker molecules, which may be advantageously used in accordance with the invention include, for example, aryl acetylene, ethylene glycol ohgomers containing from 2 to 10 monomer units, diammes, diacids, ammo acids, and combinations thereof. Other linker molecules may be used in accordance with the different embodiments of the present invention and will be recognized by those skilled m the art in light of this disclosure.

According to another preferred embodiment, linker molecules may be provided with a cleavable group at an intermediate position, which group can be cleaved with an electrochemically generated reagent. This group is preferably cleaved with a reagent different from the reagent(s) used to remove the protective groups. This enables removal of the various synthesized polymers or nucleic acid sequences following completion of the synthesis by way of electrochemically generated reagents In particular, derivatives of the acid labile 4,4'-dιmethyoxytπtyl molecules with an exocychc active ester can be used m accordance with the present invention. These linker molecules can be obtained from Perseptive Biosystems, Frammgham, Massachusetts. More preferably, N- succιnιmιdyl-4-[bιs-(4-methoxyphenyl)-chloromethyl]-benzoate is used as a cleavable linker molecule during DNA synthesis. The synthesis and use of this molecule is described m A Versatile Acid-Labile Linker for Modification of Synthetic Biomolecules, by Brian D Gildea, James M. Coull and Hubert Koester, Tetrahedron Letters. Volume 31, No 49, pgs 7095-7098 (1990) Alternatively, other manners of cleaving can be used over the entire array at the same time, such as chemical reagents, light or heat.

The use of cleavable linker groups affords dissociation or separation of synthesized molecules, e.g., polymers or ammo acid sequences, from the electrode array at any desired time. This dissociation allows transfer of the, for example, synthesized polymer or ammo acid sequence, to another electrode array or to a second substrate. Obviously, those skilled in the art can contemplate several uses for transferring the molecules synthesized on the original electrode to a second substrate.

The arrays need not be in any specific shape, that is, the electrodes need not be in a square matrix shape. Contemplated electrode array geometries include: squares; rectangles; rectilinear and hexagonal grid arrays with any sort of polygon boundary; concentric circle grid geometries wherein the electrodes form concentric circles about a common center, and which may be bounded by an arbitrary polygon; and fractal grid array geometries having electrodes with the same or different diameters. Interlaced electrodes may also be used in accordance with the present invention. Preferably, however, the array of electrodes contains at least 100 electrodes in a 10x10 matrix. One embodiment of a substrate that may be used in accordance with the present invention having a 10x10 matrix of electrodes.

More preferably, the array of electrodes contains at least 400 electrodes in, for example, an at least 20x20 matrix. Even more preferably, the array contains at least 1024 or 2048 electrodes in, for example, an at least 64x32 matrix, and still more preferably, the array contains at least 204,800 electrodes in, for example, an at least 640x320 array. Other sized arrays that may be used in accordance with the present invention will be readily apparent to those of skill in the art upon review of this disclosure.

Electrode arrays containing electrodes ranging in diameter from approximately less than 1 micron to approximately 100 microns (0.1 millimeters) are advantageously used in accordance with the present invention. Further, electrode arrays having a distance of approximately 10-1000 microns from center to center of the electrodes, regardless of the electrode diameter, are advantageously used in accordance with the present invention More preferably, a distance of 50-100 microns exists between the centers of two neighboring electrodes

The electrodes may be flush with the surface of the substrate However, in accordance with a preferred embodiment of the present invention, the electrodes are hemisphere shaped, rather than flat disks More specifically, the profile of the hemisphere shaped electrodes is represented by an arctangent function that looks like a hemisphere Those skilled the art will be familiar with electrodes of this shape Hemisphere shaped electrodes help assure that the electric potential is constant across the radial profile of the electrode That is, hemisphere shaped electrodes help assure that the electric potential is not larger near the edge of the electrode than in the middle of the electrode, thus assuring that the generation of electrochemical reagents occurs at the same rate at all parts of the electrode

Electrodes that may be used in accordance with the invention may be composed of, but are not limited to, noble metals such as indium and/or platinum, and other metals, such as, palladium, gold, silver, copper, mercury, nickel, zinc, titanium, tungsten, aluminum, as well as alloys of various metals, and other conducting materials, such as, carbon, including glassy carbon, reticulated vitreous carbon, basal plane graphite, edge plane graphite and graphite Doped oxides such as indium tin oxide, and semiconductors such as silicon oxide and gallium arsenide are also contemplated Additionally, the electrodes may be composed of conducting polymers, metal doped polymers, conducting ceramics and conducting clays Among the noble metals, platinum and palladium are especially preferred because of the advantageous properties associated with their ability to absorb hydrogen, i e , their ability to be "preloaded" with hydrogen before being used m the methods of the invention

In accordance with other preferred embodiments of the present invention, one or more of the electrodes are proximate to a "getter" structure Preferably the "getter" structure comprises a second electrode The second electrode may be of any shape or size However, it may function to scavenge electrochemically generated reagents alone or in conjunction with a scavenging solution and/or a buffering solution or it may function to reduce or eliminate diffusion of ions into nearby electric sources such as semiconductor circuitry Such second electrodes may be made of the same material as the selected electrodes discussed above

The electrode(s) used m accordance with the invention may be connected to an electric source m any known manner Preferred ways of connecting the electrodes to the electric source include CMOS switching circuitry, radio and microwave frequency addressable switches, light addressable switches, and direct connection from an electrode to a bond pad on the perimeter of a semiconductor chip The placement of a "getter" structure m accordance with the description set forth above effectively prolongs the life of a semiconductor chip thereby making such a connection particulary advantageous CMOS switching circuitry involves the connection of each of the electrodes to a CMOS transistor switch The switch is accessed by sending an electronic address signal down a common bus to SRAM (static random access memory) circuitry associated with each electrode. When the switch is "on", the electrode is connected to an electric source This is a preferred mode of operation.

Radio and microwave frequency addressable switches involve the electrodes being switched by a RF or microwave signal. This allows the sw itches to be thrown both with and/or without using switching logic The switches can be tuned to receive a particular frequency or modulation frequency and switch without switching logic Alternatively, the switches can use both methods.

Light addressable switches are switched by light. In this method, the electrodes can also be switched with and without switching logic The light signal can be spatially localized to afford switching without switching logic This is accomplished, for example, by scanning a laser beam over the electrode array; the electrode being switched each time the laser illuminates it. Alternatively, the whole array can be flood illuminated and the light signal can be temporally modulated to generate a coded signal. However, switching logic is required for flood illumination.

One can also perform a type of light addressable switching in an indirect way. In this method, the electrodes are formed from semiconductor materials. The semiconductor electrodes are then biased below their threshold voltage At sufficiently low biases, there is no electrochemistry occurring because the electrons do not have enough energy to overcome the band gap. The electrodes that are "on" will already have been switched on by another method. When the electrodes are illuminated, the electrons will acquire enough energy from the light to overcome the band gap and cause electrochemistry to occur.

Thus, an array of electrodes can be poised to perform electrochemistry whenever they are illuminated. With this method, the whole array can be flood illuminated or each electrode can be illuminated separately. This technique is useful for very rapid pulsing of the electrochemistry without the need for fast switching electronics. Direct connection from an electrode to a bond pad on the perimeter of the semiconductor chip is another possibility, although this method of connection could limit the density of the array.

Electrochemical generation of the desired type of chemical species requires that the electric potential of each electrode have a certain minimum value. That is to say, a certain minimum potential is necessary, which may be achieved by specifying either the voltage or the current. Thus, there are two ways to achieve the necessary minimum potential at each electrode: either the voltage may be specified at the necessary value or the current can be determined such that it is sufficient to accommodate the necessary voltage The necessary minimum potential value will be determined by the type of chemical reagent chosen to be generated One skilled in the art can easily determine the necessary voltage and/or current to be used based on the chemical species desired. The maximum value of potential that can be used is also determined by the chemical species desired If the maximum value of potential associated with the desired chemical species is exceeded, undesired chemical species may be resultantly produced

By including a preconstructed multidentate hgand m the form of a modified ammo acid on the surface of the array, a library of chelators can be constructed using standard peptide coupling techniques to give an extremely diverse population of chelating compounds These chelating compounds are predisposed to bind metal ions The specificity of such binding is determined by the random structure of the peptide created, and hence the availability of the binding functionality with the peptide system In some embodiments, the peptide backbone may position two or more modified chelating groups in such proximity that their concerted actions may be used to enhance binding affinity

In a third aspect, the present invention features methods for designing a chelating agent suitable for one or more particular ions certain conditions. The methods of the present invention comprise the steps of (a) constructing a combinatorial array of chelator molecules, preferably attached to a peptide chain linked to an array; (b) screening the array to determine which chelator molecules are optimal for removing one or more ions a given environment; (c) constructing a second array having these optimal chelator molecules, preferably attached to a peptide chain linked to the array; and (d) exposing the array of (c) to an environment having the ion to be removed.

The methods of the present invention feature constructing a combinatorial library of chelator molecules. Such a combinatorial library may be produced on an array or on beads well known m the art. Preferably, the electrochemical synthesis methods described m detail according to the first aspect of the invention are used to prepare an array of peptides wherein one or more of the peptides contain at least one chelating or multidentate hgand or moiety. The array comprising chelator molecules may then be screened to determine which chelators are best suited to removing or extracting a particular metal from a specific environment Those compounds that are found to be optimal metal chelators may then be placed on a diagnostic array. This array of chelators may then be exposed environment from which metal ions are to be extracted.

Those compounds that are found to be the best metal chelators from the first synthesized array may be placed on a diagnostic array. Such an array may contain virtually any desired number of chelators, for example 1 or a million different chelating agents. The second array containing optimal chelator molecules, ligands or moieties may then be exposed to an environment from which metal ions are to be extracted Analysis of this array may reveal those chelators that are most successful in removing a desired metal ion. These optimized chelators may then be produced for selectively removing a desired ion or metal material from a specific environment.

Once a suitable chelator has been found with tight and selective binding for a metal or ion to be removed from a particular environment, it may be immobilized on a stable resin to create a material capable of removing selected contaminating metals from the environment as depicted in Figure 9. Once the resin has absorbed a maximum load of the desired ion, it may be removed and replaced with fresh resin. The contaminated resin may be processed for disposal as a more compact form of waste. Alternatively, the resin may be regenerated for further use and the now concentrated and purified contaminant species may be disposed of as a concentrated waste or recycled for a more useful purpose. When selective and tight binding chelators are identified for each unwanted species in a waste stream, resins may remove the contaminating species and the remaining waste remediated in a more facile manner.

A peptide-based backbone is preferably provided on the arrays according to the present invention to give the chelating molecules of the present invention greater freedom of motion. Amino acid side chains may position themselves as necessary for optimal binding. Further, known chelating groups in the form of modified amino acids may be used to help reduce the loss of entropy on binding of the metal ion. Exemplary modifications to amino acids are illustrated in Figure 10 where the amino acids cysteine and serine are modified to include attached binding groups such as iminodiacetic acid and diethylenetriamine that may be incorporated into a peptide-based chelating molecule.

An initial exploratory library of chelators according to the present invention may be produced by either chip-based chemistry or by traditional bead-based chemistry. Once this library of chelators has been synthesized and screened, those chelators demonstrating optimal chelating abilities, i.e. the strongest and most selective binding, are normally selected for several "best candidate" libraries. These optimal chelator libraries are normally synthesized on a microelectrode array chip according to the present invention. The chip-based libraries of chelators are used to select the most appropriate chelators for isolating contaminants from any specific remediation site. Those chelators such as exemplified in Figure 11 found to be most applicable for specific chemical environments may be produced in bulk for large-scale remediation efforts. Figure 12 demonstrates an exemplary array of chelating molecules according to the present invention.

The method of screening chip-based combinatorial chelator libraries according to the present invention is illustrated in Figure 13. Here, a library on an array is exposed to a waste stream that is a remediation target. Electrochemical high throughput screening of the chelator library enables rapid identification of one or more appropriate chelator molecules for the targeted waste stream.

Anticipated benefits of the present invention include the separation of wastes into compact homogeneous forms that will significantly simplify its disposal or recycling into further useful forms Any producer of dilute waste containing metallic ions should benefit from the ability to remove selected contaminants from their waste streams.

In a fourth aspect, the present invention provides a method for chelating one or more ions present in a sample comprising the step of placing a sample, preferably a liquid or gas sample, in contact with an array having at least one and preferably a plurality of ammo acids attached thereto wherein one or more amino acids is modified to include a chelating or multidentate hgand. Preferred chelating or multidentate ligands may be selected from the group consisting of a polyamme, a crown ether of any size in which all hetero atoms consist of O, NH or S in any combination, acetoacetamide. acetoacetic acid, a porphyπn or a mixture thereof

The methods of the present invention are especially useful for separating wastes into compact homogeneous forms. This significantly simplifies disposing of or recycling the waste into useful forms. Producers of radioactive wastes containing metallic ions dilute concentrations will benefit from the methods of the present invention providing the ability to remove selected contaminants from waste streams. Additionally chelators derived from the methods of the present invention may be used for extracting valuable ions from dilute sources such as ocean water where considerable resources are available in extremely dilute concentrations.

The methods of the present invention are particularly applicable for remediating sites contaminated by radioactive waste. Contaminating species are often present at very high dilutions in waste streams. Moreover, several different radioactive moieties are frequently present in a sample. Removing radioactive species selectively from bulk waste material enables more facile disposal and recycling. Isolated radioactive species may be more compact and amenable to long-term storage than bulk waste. Alternatively, isolated radioactive waste species may be recycled into usable forms.

Sequestering of radioactive contaminants by a chelating agent may be visualized by an array of scintillating optical fibers, x-ray film, etc. However, distinguishing between different radioactive species in a high throughput system by these methods is problematic. It is also desirable to perform initial screening of candidate chelators using isotopes of metal ions that are not radioactive. This substantially reduces the expense of handling and disposing of assay chemicals. Electrochemistry provides a natural assay technology for chelator candidates arrayed on microelectrodes The redox potentials of most chelated lanthamde and actimde complexes are sufficiently well separated that they can be distinguished electrochemically However, the complexes may be immobilized within a membrane and cannot contact the electrodes directly Redox mediators are a well-known method for ferrying electrons to species immobilized in polymer films overlaying electrodes A series of redox mediators can be used to perform indirect voltammetry on immobilized chelator complexes

Electrochemistry may provide quantitative information regarding the identity of metal ions complexed by chelator complexes Further, electrochemical measurements on microelectrode array chips may be performed in parallel on any number of selected electrodes This enables facile automation of high throughput screening for chelator complexes according to the present invention

Using combinatorial technology for synthesizing potential chelating species allows rapid construction of large libraries with many potential chelator candidates High throughput screening of these libraries allows assessing compounds for their ability to bind contaminants in any selected chemical environment With a combinatorial library of sufficient size, there is a high probably that chelators may be identified having optimal chelating abilities in any specific situation Materials removed from a waste stream contacted with such a chelating array are normally highly enriched and are suited for converting into a useful form

The use of a combinatorial chelator array is particularly beneficial m a wide variety of applications One method of treating exposure to plutonium is to effect its removal by the use of chelating agents which bind to the plutonium ion and are then excreted A chelator array according to the present invention may be used to develop chelators having a stronger binding affinity for plutonium thereby making them more effective An array of chelators may be prepared and exposed to a plutonium containing solution and the resulting capture of plutonium on the array may be quantitated using either scintillation counting or by exposure of shielded photographic film Those chelators identified as having the highest binding may be used as the starting points for further investigation in additional arrays After several rounds of optimization by this process the best binding chelators may be synthesized using classic methodology to produce quantities of material for testing

Exposure to mercury and organomercureal compounds is currently treated using chelation therapy An improved drug for removing mercury could be created using the present invention An array of chelators may be prepared and exposed to a mercury containing solution The mercury captured by such exposure may be quantitated using mass spectral analysis of the resulting membrane Those chelators identified as having the highest binding may be used as the starting points for further investigation in additional arrays After one or several rounds of optimization by this process the best binding chelators may be synthesized using classic methodology to produce quantities of material for testing.

DESCRIPTIO OF THE PREFERRED EMBODIMENTS

The following are provided purely by way of example and are not intended to limit the scope of the present invention

EXAMPLE 1 Description and Preparation of the electrode array chips

The chips prepared and used in the present invention are rectangular devices with a 16 (m the x-direction) by 64 ( the y-direction) array of 100 micron diameter platinum electrodes The total number of electrodes m these arrays was 1024. The dimensions of the chips were approximately 0.5 cm (x- direction) by 2.3 cm (y-direction), and the total surface area of the chips was approximately 1 square centimeter. The electrodes each array were approximately 250 microns apart in the x-direction and approximately 350 microns apart in the y- direction, measured from the center of the electrodes.

Each electrode in the array was capable of being addressed independently using an SRAM cell (static random access memory), a standard art-recognized way to address independently electric circuitry m an array. The SRAM cell was located next to the electrodes m the electrical circuitry associated with electrode. Each electrode in the array had four separate switchable voltage lines that attached to it, allowing each electrode in the array to be switched independently from one voltage line to another. The voltage was arbitrary and was set by an external voltage source.

The chips used were additionally 13 electrodes on the side of the chips that were hard wired to bond pads, meaning they were not switchable or independently addressable as were the electrodes in the 16x64 array. These 13 electrodes had no circuitry associated with them except for a single voltage line, and thus allowed protocols to be run on them without engaging the associated electrode array. These 13 electrodes were 100 microns in diameter and were spaced differently from the electrodes in the array.

The chips were made by a 3 micron process using hybrid digital/analog very large scale integration (VLSI). One skilled in the art would be familiar with such a process and could easily prepare a chip for use in accordance with the present invention. See, Mead, C, Analog VLSI and Neural Systems. AddisonWesley (1989). The circuitry used was CMOS (complimentary metal-oxide silicon) based and is also well known to those of ordinary skill in the art.

The chips were controlled by at least one Advantech PCL-812 digital I/O card (in the computer) that was driven by a Pentium based personal computer These digital I/O cards can be obtained from Cyber Research, Branford, Connecticut Preferably the chip is connected through interface hardware, i e , an interface card, to the I/O card. The software for driving the I/O card can easily be written by one of ordinary skill in the art. DC voltage for powering the chips was provided by the PCL- 812 and/or a Hewlett-Packard E3612A DC power supply. Voltage for the electrodes was supplied by the PCL-812 card and/or by an external Keithley 2400 source- measure unit.

The electrode array chips were designed so that the bond pads for all of the on- chip circuitry were located at one end of the long side of the chips. The chips were attached to a standard 121 pin PGA (pin grid array) package that had been sawn m half so that approximately 2 cm of the chip extended out from the end. analogous to a diving board PGA packages can be obtained from Spectrum Semiconductor Materials, San Jose, California. Connecting wires ran between the bond pads on the chip and the contacts (bond pads) on the PGA package. The bond pads on the chip, the connecting wires, and the contacts on the PGA package were covered with epoxy for protection and insulation. The section of the chips that extended into the air contained the electrode array and was not covered by epoxy. This section of the chips was available for dipping into solutions of interest for chemical synthesis at the electrodes at the surface of the chip. One of ordinary skill in the art could easily set up and design chips appropriate for use m accordance with the present invention.

Preparation of the chip for attachment of molecules

To enable the attachment of molecules, in particular trityl linker molecules, to the surface of the electrode array chip for synthesis and/or deprotection proximate the electrodes, the chip was coated/modified with an overlaying membrane of a polysacchaπde-based material. Specifically, a polygalactoside was used as the overlaying membrane material m this example. The polygalactoside membrane was dip coated onto the chip. However, dipping or coating according to any method known to one of ordinary skill in the art would be acceptable.

Attachment of the trityl linker molecules

Once the electrode array chip was coated with the polysacchaπde membrane, the trityl linker molecules were attached to the chip. The trityl linker molecule used for this example was a modified 4,4'-dιmethoxytπtyl molecule with an exocychc active ester, specifically the molecule was N-succιnιmιdyl-4[bιs-(4-methoxyphenyl)- chloromethyl]-benzoate. The synthesis and use of this molecule is described in A Versatile Acid-Labile Linker for Modification of Synthetic Biomolecules, by Brian D. Gildea, James M. Coull and Hubert Koester, Tetrahedron Letters. Volume 31, No. 49, pgs 7095-7098 (1990).

The trityl linker molecules were attached to the polysacchaπde membrane via immersion of the polysacchaπde membrane coated chip in an acetonitπle solution containing

0.5M of tertbutyl ammonium perchlorate, 0.75M of 2,4,6-colhdine and 0.2M of the trityl linker The immersion of the polysacchaπde membrane coated chip the DMF linker solution lasted for 30 minutes at ambient temperature. The trityl linker coated chip was then washed with DMF to remove any remaining reactants. Next, the trityl linker coated chip was washed m an aqueous 0.1 M sodium phosphate buffer that was adjusted to pH 8 0. and dried

Selective Deprotection

To perform the selective deprotection step, the prepared chip was immersed m a 0.05M aqueous sodium phosphate buffer solution to enable electrochemical generation of reagents. A voltage difference of 2.8 volts was applied to select electrodes (alternating in a checkerboard pattern) for approximately 10 minutes, causing protons to be generated electrochemically at the anodes.

After the protons were electrochemically generated at the anodes, the anodes became dark because the trityl linker previously bound proximate to. the anodes dissociated from the anodes and the fluorescent labeled streptavidm molecules were washed away. The extent to which this occurred at the anodes and not at the cathodes m the checkerboard pattern, is a measure of the chemical crosstalk occurring between the electrodes m the array. That is, if chemical crosstalk were occurring, the cathodes would also be dark because the protons would have migrated and dissociated the trityl linkers at the cathodes.

Thus, under epifluorescent microscopy, the bright electrodes (cathodes) indicate the presence of a Texas Red labeled streptavidm molecule bound to a linker molecule at the electrode and the dark electrodes (anodes) indicate the lack of a Texas Red labeled streptavidm molecule bound to a linker molecule at the electrode.

Results

Selective deprotection was achieved using the process of the present invention. A repeating checkerboard pattern was produced, exemplifying that the process of the present invention achieved localization of the protons generated at the anodes and prevented migration of these protons to the cathodes. The dark areas (anodes) are clearly defined and distinguished from the also clearly defined bright areas (cathodes). The clearly demarcated checkerboard pattern shown m the photomicrographs indicates that no, or very little, chemical cross talk occurred during the deprotection step.

EXAMPLE 2 Construction of a peptide based chip

A chip is coated in agarose and croshnked for stabilization This chip is placed in a solution of linker carbonate #1, methanol, DMF vitamin B12 and TBAN and a full checker board pattern is applied using 1.5 to 2.5 volts so that a pattern of linker immobilized spots is produced The chip is deprotected selectively by immersing it in a solution of methanol and Piperidmehydrooxalate A current is passed at 2 volts to generate a base which deprotonated the piperidme hydrochlonde producing piperidme which consequently removed the fmoc protecting group on the immobilized linker m a controlled pattern This leaves a chip patterned with amine groups. The chip is immersed in a DMF solution of an activated ammo acid (FMOC protected- on the amine side and Pentafluorophenol activated on the acid side) and dnsopropyl ethyl amme After coupling has occurred on the exposed ammes, a new portion of the chip is deprotected and the chip is exposed to a new ammo acid Consecutive layering of ammo acids across varying portions of the chip produces a surface patterned with peptides of varying ammo acid sequence and structure At any point in the process, a modified ammo acid containing a chelating functional group may be incorporated into the growing peptide sequence Figure 14 exemplifies the inclusion of a modified amino acid Alternatively or additionally, the terminal ammo acid may be modified by exposing it to a dianhydπde such as exemplified herein. The chip may be finally deprotected by exposing it to aqueous piperidme then acid (for example, TFA) to yield the active membrane bound peptide chelator ready for on chip testing

The array may then be immersed in a solution of the ion to be analyzed. In the case of an amine based array, such as created by the inclusion of compound 2 a plutonium solution may be used Each peptide provides a unique microenvironment for the chelator potentially adding additional strength of chelation to the system. The chelator corresponding to the most radioactive spot as determined by exposure of photographic film exemplifies the best plutonium chelator the array and provides the lead compound for further research.

A large variety of chelator molecules may be used as are known to those of skill in the art Exemplary chelator molecules are set forth in Figure 6, compounds 1-7 For instance, compound 1 could be used as a chelator molecule whereby it could be connected via attachment to one of the aromatic rings Compound 2 could be substituted for EDTA anhydride, or compound 3 could be used with a coupling reagent for similar effect. Compound 4 represents a bmaphthyl chelator, it could be attached either at one of the aromatic rings or via one of the R groups. Compound 5 represents one of many polyhydroxylated species which could be attached for additional functionality. Compound 6 represents an aceto acetate group. An especially preferred R constituent could be the amine of the growing peptide or a carbon based attachment point to the system Compound 7 is similar having an attachment position in the middle providing a malonate derivative Peptide — NH₂

y

Any of many coupling reagents Chelator

H₂ —

Ready for use as a modified amino acic The use of compounds such as compound 6 are exemplified. First the growing peptide may be end terminal capped using aceto acetate to install the aceto acetate group at the end of the peptide. Alternatively lysine can be modified and protected as shown to produce an aceto acetate modified side chain. This modified amino acid may then be substituted in the main chain just as any other amino acid in the synthesis methods described herein.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

WHAT IS CLAIMED IS:

1 A method for making an array of one or more metal chelators comprising the steps of

(a) providing a substrate having at its surface at least one electrode that is proximate to at least one molecule bearing at least one protected chemical functional group,

(b) applying a potential to the electrode sufficient to generate electrochemical reagents capable of deprotecting at least one of the protected chemical functional groups of the molecule, and

(c) bonding the deprotected chemical functional group with a modified amino acid having a chelating moiety.

2 A method for making an array of one or more metal chelators comprising the steps of.

(a) placing a buffering or scavenging solution in contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto,

(b) selectively deprotecting at least one protected chemical functional group on at least one of the molecules;

(c) bonding a first monomer having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule,

(d) selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group;

(e) bonding a second monomer having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule; and

(f) repeating the selective deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule and the subsequent bonding of an additional monomer to the deprotected chemical functional group until at least two separate polymers containing a chelating hgand of desired length are formed on the substrate surface

3 A method for making an array of one or more metal chelators comprising the steps of:

(a) placing a buffering or scavenging solution in contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto, (b) selectively deprotecting at least one protected chemical functional group on at least one of the molecules,

(c) bonding a first monomer having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule,

(d) selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group;

(e) bonding a second monomer having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule; and

(f) repeating the selective deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule and the subsequent bonding of an additional monomer to the deprotected chemical functional group until at least two separate polymers containing a chelating hgand of desired length are formed on the substrate surface

4. The method according to claim 1 wherein the metal chelators are selected from the group consisting of a polyamme, a crown ether of any size in which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyrm or a mixture thereof

5. An array having at least one amino acid attached wherein the ammo acid is modified to include a chelating or multidentate hgand.

6. An array according to claim 5 having a plurality of ammo acids

7. An array according to claim 5 wherein the chelating or multidentate ligands are selected from the group consisting of a polyamme, a crown ether of any size m which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyrm or a mixture thereof

8. An array according to claim 5 wherein a plurality of amino acids are attached to the array by electrochemical means.

9. A method for obtaining a chelating agent suitable for one or more particular ions m certain conditions comprising the steps of

(a) constructing a combinatorial library of chelator molecules;

(b) screening the combinatorial library of chelator molecules to determine optimal chelators; (c) constructing an array of optimal chelators screened from (b)

10. The method of claim 9 wherein the combinatorial library of chelating agents is constructed by electrochemical means on an array.

1 1. The method of claim 9 wherein the combinatorial library of chelating agents comprises chelating ligands bound to a modified amino acid.

12. A method for chelating one or more ions present m a sample comprising the step of placing a sample in contact with an array having at least one amino acid attached wherein at least one ammo acid has been modified to include at least one chelating or multidentate hgand.

13. A method according to claim 12 wherein the chelating or multidentate hgand is selected from the group consisting of a polyamme, a crown ether of any size in which all hetero atoms consist of O, NH or S in any combination, acetoacetamide, acetoacetic acid, a porphyrm or a mixture thereof.