WO1994015628A1

WO1994015628A1 - Electrically conducting synthetic peptide complexes

Info

Publication number: WO1994015628A1
Application number: PCT/US1994/000215
Authority: WO
Inventors: Dan E. Robertson; Ramy S. Farid; William F. Degrado; P. Leslie Dutton
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 1993-01-12
Filing date: 1994-01-06
Publication date: 1994-07-21
Also published as: IL108309A0; AU6084194A

Abstract

Electrically conducting synthetic peptide complexes and methods for their preparation. The synthetic peptide complexes comprise first and second peptides which are spaced in parallel relationship. The peptides possess a helical structure and are bound by a plurality of complexing agents. The complexing agents have redox potential asymmetry which provides the synthetic complex with an electrical bias. The synthetic peptide complexes are particularly useful in electronic devices, including molecular electronic devices.

Description

ELECTRICALLY CONDUCTING SYNTHETIC PEPTIDE COMPLEXES

Field of the Invention This invention relates to electrically conducting materials. More specifically, this invention relates to electrically conducting synthetic peptide complexes, to methods for preparing such complexes, and to electronic devices which incorporate the complexes.

Known electrically conductive materials include metals, such as silver, copper and aluminum; certain types of hydrocarbon-based polymers, for example, polyacetylene-type resins; and certain types of ceramics, for example, titanium suboxide-type materials. Such conducting materials can be formed into various devices including wires, electrodes, films, transistors, diodes, rectifiers, integrated circuits, resistors and capacitors. Such devices can be combined to form circuits, which in turn, can be combined to form systems, such as those used in television sets, clocks, radios and countless other products. It has been recognized also that there are naturally occurring biological materials that are electrically conductive. Thus, it is known that certain naturally occurring proteins possess a variety of physical properties that enable them to participate in respiratory and photosynthetic electron transfer reactions. For example, cytochrome bCj complexes are redox enzymes which are capable of catalyzing the oxidation of hydroquinone and reduction of cytochrome c. The cytochrome be, complexes operate to separate charge across a biological membrane, generating strong electric fields.

Based on their physical properties, proteins that are capable of performing electron transport and photochemical processes are considered to be potentially useful in the construction of molecular electronic devices

(MEDs) , that is: (A) devices that use materials whose unique properties result from their molecular structure and which are capable of sensing, responding to, or recording electrical or physical stimuli; and (B) devices that embody the concept that individual molecules are functionally capable of responding to stimuli and that they have the potential to be interconnected in order to functionally replicate an electronic circuit. See, e.g., Sligar et al.. Current Opinion and Structural Biology, 2 , 587-592 (1992); Sligar et al., Current Opinion in Biotechnology, 3, 388-394 (1992) , which are hereby incorporated by reference.

Despite the powerful techniques available of modifying natural protein through recombinant DNA technology, the realization of the potential usefulness of such proteins has been an unfulfilled challenge for a variety of reasons. For example, naturally occurring proteins generally possess protein domains which may perform a biological function, but which may not contribute, and may actually detract from, the desirable electrical properties of the protein. Although the practical application of proteins and molecular electronics devices is still in its infancy, it is apparent that protein molecules represent the ultimate miniaturization possible in individual electronic devices.

Summary of the Invention

In accordance with the teachings of this invention, a process for preparing an electrically conducting synthetic peptide complex is provided. This process involves providing first and second peptides, each of which have a helical structure. The first and second peptides are combined with a plurality of complexing agents. The complexing agents bind with the peptides to provide the synthetic peptide complex which has an electrical bias.

In preferred form, the processes of this invention involve linking together the first and second peptides to form dimeric peptides which are then combined with a metal- containing complexing agent.

In other embodiments, a long peptide can be folded and complexed to itself with these agents. Accordingly, electrically conducting organic complexes can be produced synthetically and efficiently without recombinant DNA techniques. The complex can be tuned, using different combinations of amino acid side chains, complexing amino acid residues, and complexing agents to provide all types of micro-electronic devices. For example, the complexing agents can be selected to provide electrical bias, like a diode, or temperature independent range. All of these devices can be synthetically manufactured without interfering protein domains found in naturally-occurring peptide chains.

In more detailed embodiments of this invention, the conducting synthetic complex comprises peptides which are spaced in parallel relationship. The peptides have a helical structure and are bound by a plurality of complexing agents. The complexing agents are characterized by redox potential asymmetry which provides the synthetic complex with a preselected electrical bias or property.

In preferred form, the first and second peptides comprise α-helices and are linked together at the amino terminus. In addition, the complexing agents preferably comprise compounds which are capable of forming complexes with side chains of amino acid residues.

Also in accordance with this invention, the electrically conducting synthetic peptide complex includes a preferred amino acid arrangements, including the following amino acid sequence: Cys-Gly-Gly-Gly-Glu-Leu-Trp-Lys-Leu-His- Glu-Glu-Leu-Leu-Lys-Lys-Phe-Glu-Glu-Leu-Leu-Lys-Leu-His-Glu- Glu-Arg-Leu-Lys-Lys-Leu. The pair of peptides are linked together at residue 1 (cysteine) . The peptide complex also comprises two iron protoporphyrin IX compounds. The first of the iron protoporphyrin IX compounds is complexed between the residues 10 (histidine) and the second of the iron protoporphyrin IX compounds is complexed between the residues

24 (histidine) .

This invention also provides an electrically conducting synthetic peptide complex comprising the following formula.

R-C(=0)-CH(NH₂)-CH₂-S-S-CH₂-CH(NH₂)-C(=0)-R, I

R and R_j are each independently peptides having the following amino acid sequence: -Gly-Gly-Gly-Glu-Leu-Trp-Lys-

* Leu-His-Glu-Glu-Leu-Leu-Lys-Lys-Phe-Glu-Glu-Leu-Leu-Lys-Leu-

# His-Glu-Glu-Arg-Leu-Lys-Lys-Leu. This peptide complex also comprises two iron protoporphyrin IX compounds. The first of the iron protoporphyrin IX compounds is complexed between the *

His residues of each of the R and R_ peptides and the second of the iron

# protoporphyrin IX compounds complexed between the His residues of each of R and Rj.

Definitions

As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. "Alkyl", either alone or with various substituents defined herein, means a saturated aliphatic hydrocarbon, either branched- or straight-chained. A "lower alkyl" is preferred, having about one to about six carbon atoms.

Examples of alkyl include methyl, ethyl, .n-propyl, isopropyl, butyl, sec-butyl, t-butyl, amyl and hexyl.

"Aryl" means an unsaturated ring system characteristic of benzene. Preferred aryl groups include ring systems of from about six to about 10 carbon atoms, and include phenyl, naphthyl, and phenanthryl.

"Alkenyl" refers to a hydrocarbon having at least one point of unsaturation and may be branched- or straight- chained. A "lower alkenyl" is preferred having about two to about six carbon atoms. Examples of alkenyl include vinyl, allyl, ethynyl and isopropenyl.

"Aralkyl" means an alkyl group substituted by an aryl radical. The preferred aralkyl groups are benzyl or phenethyl.

"Alkylcarboxy" refers to alkyl substituted carboxy groups.

"Halo" means a halogen. Preferred halogens include chloride, bromide and fluoride. "Peptide" means a polymer of amino acids and includes oligopeptides and polypeptides. "Di eric" refers to a covalent association of two peptides.

"Side chain" means the group designated as "R" in the following generic formula for amino acids: H₂N-CH(R)-COOH.

Table 1 shows the standard three letter designations and single letter for amino acids as used in the application.

"Neutral amino acid residue", as used herein, means any amino acid residue which is generally free of positively and/or negatively charged side chains.

For the purpose of illustrating the invention, there are shown in the drawings forms which are presently preferred; it being understood, however, that this invention is not limited to the precise arrangement and instrumentality shown.

Figure 1 is a schematic representation of a synthetic peptide complex in accordance with a preferred embodiment of the present invention.

Detailed Description of the Invention Methods for preparing an electrically conducting synthetic peptide complex and peptide complexes produced thereby are provided by this invention. The processes involve providing first and second peptides having a helical structure. These peptides are combined with a plurality of complexing agents. The complexing agents bind with the peptides to provide the synthetic peptide complex with an electrical bias.

The peptide complexes of the present invention are particularly suitable for use in molecular electronic devices (MEDs) . The synthetic peptide complexes of the present invention provide nanometer scale, single molecule electronic units for use in various electronic devices.

As indicated above, the electrically conducting synthetic peptide complexes of the present invention comprise first and second peptides. It is generally understood that the amino acid sequence of each of the first and second peptides may be the same or different. In addition, the length of each of the first and second peptides may be the same or different.

The peptides of this invention can take on a number of secondary structures, including, for example, 3_I0, α and n helices and 0-sheets. Preferably, the helical peptides possess an α-helical structure.

In accordance with preferred embodiments of the present invention, the preferred helical peptides are spaced in parallel relationship relative to each other. In certain preferred embodiments, the orientation of the net dipole moments of the helical peptides are substantially the same. In certain other embodiments, the orientation of the net dipole moments of the helical peptides are substantially different. The spaced parallel relationship of a preferred helical peptide arrangement is depicted generally in Figure 1.

It is contemplated that the helical peptides may possess a first face comprised of hydrophilic residues and a second face comprised of hydrophobic residues. In certain embodiments, it is contemplated that the helical peptides will orient themselves in a parallel relationship wherein the hydrophobic residues are in facing relationship and the hydrophilic residues are in opposing relationship when dissolved in polar solvents. Such solvents include, for example, water.

In certain other embodiments, it is contemplated that the helical peptides will orient themselves in a parallel relationship wherein the hydrophobic residues are in facing relationship and the hydrophobic residues are in opposing relationship when dissolved in non-polar solvents or incorporated into lipid bilayer. Such solvents include, for example, hexane.

In those embodiments wherein the hydrophobic residues are in facing relationship and the hydrophobic residues are in opposing relationship, the preferred amino acid sequence is Cys-Gly-Gly-Gly-Ala-Leu-Trp-Aib-Leu-His-Leu- Ala-Leu-Leu-Aib-Leu-Phe-Ala-Leu-Leu-Leu-Aib-Ala-His-Leu-Ala- Arg-Leu-Aib-Leu-Leu, where Aib is 2-aminoisobutyric acid. As is generally understood in the art, certain amino acids are classified as being hydrophilic or hydrophobic. In preferred aspects of the present invention, the hydrophilic residues may comprise amino acids containing charged side chains, for example, lysine and glutamic acid. Also in preferred aspects of this invention, the hydrophobic residues comprise amino acids having non-polar side chains, for example, leucine and valine.

It is generally also understood in the art that certain amino acids promote the formation of α-helices and are referred to hereinafter as "helix-formers." Such helix formers include glutamine, leucine, alanine and isoleucine. Certain other amino acids are less commonly associated with helical structures, for example, proline. It is generally preferred that the amino acid residues incorporated in the helical peptides of the present invention comprise helix- formers.

The amino terminal residues of the helical peptides of this invention can include a linking agent for bonding the peptides together or a series of peptides can be bound to a substrate, such as gold, silicon, gallium or silver. When the helical peptides are linked together at their amino termini, the linking agent can contain a natural or synthetic bonding agent, such as cysteine, synthetic dipeptides or artificial amino acids. The linking agent helps the peptides polymerize or dimerize. In those embodiments wherein the amino terminal residues, most preferably the helical peptides are linked together through a cysteine residue by a cystine disulfide bond. The helical peptides of this invention also comprise at least two amino acid residues which are capable of forming complexes with the preferred complexing agents. These amino acids are referred to generally hereinafter as "complexing amino acid residues." Preferably, the complexing amino acid residues comprise heteroatoms which have lone pairs of electrons and which may form complexes with metals which are preferably present in the complexing agents. Preferably, the complexing amino acid residues comprise histidine, and more preferably the helical peptides comprise at least two complexing amino acid residues.

The accordance with preferred embodiments of this invention also can contain electron transfer modulators comprised of one or more amino acid residues of the helical peptides located between the complexing amino acid residues. These amino acid modulators tune the electrical conductivity or resistance between the complexing residues to help accelerate, maintain or discourage electron movement. Preferably, there are about 6 to about 20 amino acid residues, preferably about 10 to about 13 amino acid residues, and in certain embodiments, there are about 13 amino acid residues between the complexing amino acid residues in the helical peptides.

The amino acid modulators can be neutral, positive or negative. Preferably, each helical peptide contains at least one amino acid residue which is located between the complexing residues. These neutral amino acid residues on each helical peptide are preferably pared and located on the same face as the complexing amino acid residues. Modulator amino acid residues include those residues which comprise generally non-polar (hydrophobic) side chains, for example, phenylalanine, and amino acid residues which are generally polar but uncharged, for example, threonine. Preferably, the modulator residue is selected from the group consisting of a neutral phenylalanine, alanine, glycine, isoleucine, leucine, valine, tyrosine, serine, threonine and methionine. Preferably, the modulator residue is selected from the group consisting of a neutral phenylalanine, alanine, glycine, isoleucine, leucine and valine, with the more preferred neutral residue being phenylalanine.

The helical peptides of this invention can also optionally include at least one amino acid residue having a negatively or positively charged side chain and which is in substantially close proximity to at least one of the complexing agents. While applicants do not wish to be bound by any particular theory or theories, it is contemplated that the charged amino acid residue affects the electronic properties of the complexing agent to which the charged residue is proximate. Accordingly, in a synthetic peptide complex having a plurality of complexing agents, the complexing agent proximate to the charged residue possesses electronic properties which are different from one or more of the other complexing agents which are present in the synthetic peptide complex. The different electronic properties of the complexing agents provides the synthetic peptide complex with an electrical bias. The electrical bias is due to the redox potential asymmetry of the complexing agents. This bias can provide uni-directional current properties to the complex peptides, similar to a diode. One useful charged residue is arginine. In certain biased embodiments of this invention, about one to about four amino acid residues are employed between the charged residue and the complexing amino acid residue, and preferably, there are about two residues between the charged residue and the complexing residue.

The helical peptides of the present invention can also include additional residues for spectroscopic identification, such as a tryptophan residue, or for facilitating amino terminus bonding, such as glycine. Glycine is known to have a high degree of conformational freedom to aid in bonding to linking agents, such as cysteine. One preferred arrangement includes three contiguous glycine residues proximate to the amino terminus of each peptide helix. As noted hereinabove, the synthetic peptide complexes of the present invention preferably comprise a complexing agent. Preferred complexing agents are capable of forming complexes with compounds which comprise heteroatoms and/or compounds generally having lone pairs of electrons which are available to complex with the complexing agent. In preferred embodiments, the complexing agents comprise organometallic complexes having metal atoms which are capable of complexing with one or more of the heteroatoms and/or lone pairs of electrons. In certain embodiments, the organometallic complexing agent comprises a derivative of porphine, for example, substituted or unsubstituted porphyrins. Suitable substituents include alkyl, aryl, alkenyl, hydroxy, alkylcarboxy or halo. In addition, the porphyrins comprise a central metal atom, preferably selected from the group consisting of iron, zinc, magnesium, copper and nickel. Preferred porphyrins include, for example, chlorophylls, for example chlorophyll a and chlorophyll b, he e, octaethylporphyrin and meso-tetraphenylporphyrin. A porphyrin which is particularly suitable for use in the synthetic peptide complexes of the present invention is iron (III) protoporphyrin IX and which has the following formula.

BOOC COOH

Other porphyrins, in addition to those exemplified above, would be readily apparent in view of the present disclosure. As noted above, the number of residues between the complexing residues may vary, for example, from about six to about 20 amino acid residues. Accordingly, in those embodiments wherein the complexing agent comprises a porphyrin, the distance between the porphyrins is variable. As used herein, the "shortest edge to edge distance", as used herein, means the shortest distance between the two closest atoms which are members of conjugated ring systems of the first and second porphyrin molecules. In preferred embodiments, the shortest edge to edge distance from an edge of a first porphyrin molecule to an edge of the second porphyrin molecule should be at least sufficient so that the complexing residues do not overlap, which is about 3.5 angstroms. At this distance the complexing residues have overlapping van der Waals forces and the peptide complex acts as a super conductor having an electron flow rate of about 10¹⁰ to 10¹³ electrons per second. At the other end of the spectrum, the maximum shortest edge to edge distance should be no greater than about 35 angstroms, for distances exceeding the spacing would not provide commercially useful conductivity, i.e. the complex would behave as an effective insulator. Preferably, the shortest edge to edge distance between porphyrin molecules is about 5 to 27 angstroms, and in the preferred embodiment, about lθA which substantially corresponds to the distance observed when the complexing residues are separated by 13 amino acid residues.

In alternate embodiments, the complexing agent preferably comprises an organometallic complex which comprises a metal, preferably selected from the group consisting of ruthenium, cobalt, nickel and copper and ligands selected from the group consisting of pyridine, pyrrole, thiophene, furan and 2,2'-bipyridine. An example of such a complexing agent is ruthenium tris(bipyridyl) . In other alternate embodiments of the present invention, the complexing agent may comprise substantially small gaseous molecules, for example, CO, 0₂, HCN, H₂S and NO.

The helical peptides may be manufactured by synthesizing the peptides using standard techniques which are known to those of ordinary skill in the art. Such techniques include, for example, solid phase synthesis. Alternatively, the helical peptides may be provided by purchasing the peptides from one or more various peptide vendors. In addition, it is contemplated that the peptides of the present invention could be obtained using recombinant DNA techniques.

In particularly preferred embodiments, the methods involve linking together the pair of peptides to form di ers. The linking together may be achieved by using a variety of techniques. For example, and in those embodiments wherein the helical peptides comprise cysteine residues as the amino terminal residue, the peptides may be reacted under conditions to promote oxidation of the thiol group of the cysteine residue to provide cystine disulfide bonds. The oxidation of cysteine residues may be achieved by preparing an aqueous solution of the helical peptides. Oxidation of the cysteine residues may then be achieved by mixing and/or agitating the aqueous solution under an air atmosphere. In alternative embodiments, the helical peptides may be linked together using standard synthetic chemistry techniques. For example, the dimeric helical peptides may be prepared by providing a first peptide having a cysteine residue proximate the amino terminus with a thiol leaving group, for example, 5, 5'-dithiobis(2-nitrobenzoic acid) which is generally commercially available. A second peptide substituted with a cysteine proximate the amino terminus is then reacted together to form a disulfide bond by displacing the leaving group. The pair of helical peptides, thus formed, can then be combined with a plurality of complexing agents. The combination of the helical peptides with the complexing agents preferably involves titrating the helical peptides with two equivalents of the organometallic complexing agent.

The dimeric helical peptides may be prepared also by bridging cysteine residues with an alkyl or aryl dihalide, for example, 1,3-dinitro-4,6-difluorobenzene. In addition, the helical peptides may be linked together at their amino termini by reaction with an alkyl, aryl, aralkyl, or dicarboxylic acid, for example, 1,4-phthalic acid, and a coupling agent, such as carbodiimide.

The foregoing reactions may be carried out while the peptide is on the solid phase subsequent to removal of alpha-amino protecting groups and/or by reaction of the purified helical peptide in aqueous solution at a pH of about 7.

As indicated above, the synthetic peptide complexes of the present invention are particularly useful in various electronic devices, including MEDs. The synthetic peptide complexes which comprise two complexing agents are particularly suitable for use as integral features of sensing devices. Such sensors may be used to detect the presence of gases, for example, CO and NO. As noted above, such gases will bind with the complexing amino acid residues of the helical peptides. Sensors which comprise the synthetic peptide complexes of this invention are unique in that a pulse voltage may be applied which promotes removal of the compound which has been detected and which is bound to the complexing amino acid residue. The removability of the detected compounds is a particularly desirable feature of the sensors in that the sensor devices do not become "poisoned" . Accordingly, the sensors are extremely sensitive and long- lasting.

It is contemplated that the two complexing agents facilitate the movement of electrons in a vectorial manner within the complex. This vectorially directed charge movement permits electrical assay of the electron transfer upon the application of a stimulus. Such stimuli include (1) binding of a synthetic peptide complex with the device as a sensor; (2) light activation of electron transfer; and (3) applied voltage induced electron transfer. Detection of the electron transfer which is induced by the foregoing stimuli may be monitored by electrical signals and by color change. The synthetic peptide complexes which comprise two porphyrin molecules Flexibility in the degree of electrical contact with electrodes ranging from substantially no contact to highly efficient contact offers many analytical avenues by applying thin layer insulators, such as silicon monoxide (SiO) and polyethylene. The electrodes may comprise a variety of conducting materials, for example, gold, platinum, silver, indinium tin oxide, and mercury. Other electrode materials, in addition to those exemplified above, would be apparent in view of the present disclosure.

In addition, the synthetic peptide complexes of the present invention may be used as the basis for blood substitutes. In addition, light-activatable pigments and redox cofactors, for example, chlorophylls and ruthenium complexes ligated to the complexing amino acids, would render the present synthetic peptide complexes capable of converting light energy into electrical energy. Accordingly, the present synthetic peptide complexes may be used in solar energy devices.

It is contemplated that the conductivity of the synthetic peptide complexes of the present invention are substantially independent of temperature. In particular, the conductivity of the present synthetic peptide complexes is substantially independent of temperatures from about 4°K to about ambient temperature (298°K). This temperature independence is a desirable feature for electronic devices which operate in various temperatures, including temperatures from about 4°K to about room temperature.

Examples

Example 1

A. Synthesis of the Peptide

A peptide having the amino acid sequence Cys-Gly- Gly-Gly-Glu-Leu-Trp-Lys-Leu-His-Glu-Glu-Leu-Leu-Lys-Lys-Phe- Glu-Glu-Leu-Leu-Lys-Leu-His-Glu-Glu-Arg-Leu-Lys-Lys-Leu was prepared using standard F-moc chemistry on PAL resin by standard protocols described by the instrument manufacturer, Milligen, Inc.

B. Purification of the Peptide The peptide prepared in step A was purified by reverse-phase high pressure liquid chromatography (Vidac C-18 column; gradient mobile phase: 0.1 M triethylammonium phosphate, pH 2.2 to 70% acetonitrile/30% 0.1 M TEAP. The desired peptide eluted at 35.5 minutes. A second purification was performed using a gradient of acetonitrile from 20% to 50% containing 0.1% aqueous trifluoroacetic acid over 30 minutes. The desired peptide eluted at 24 minutes. Fractions were pooled and lyophilized. The amino acid sequence set forth in step A above was confirmed by fast atom bombardment-MS spectrometry. (M+H) ⁺ was 3,746.6 which is equal to the theoretical protonated mass for the peptide.

C. Formation of Dimer Dried peptide was dissolved in 500 millimolar (mM)

Tris(hydroxymethyl)a inoethane hydrochloride which was adjusted to a pH of about 8.9 with aqueous sodium hydroxide (1 Molar (M) ) . The final peptide concentration was about 500 micromolar (μM) in a total volume of 1 L. The solution was placed in a conical-bottomed tube with a magnetic stirring bar. The tube was placed above a magnetic stirrer and the solution was stirred under an air atmosphere. Free thiol (- SH) in the solution was measured in aliquots using Ellman's reagent (5,5'-dithio-jbis[2-nitrobenzoic acid]) as outlined in Deaken, H. et al., Biochem . J. , Vol. 89, p. 296 (1963). All cysteines were found to be oxidized and dimerization was complete within about 2.5 hours. The dimerized peptide was stored at a concentration of about 500 μM at 4°C.

D. Addition of Complexing Agent A solution of iron protoporphyrin IX chloride in dimethylsulfoxide (DMSO) was prepared at a concentration of about 1 M. Stoichiometric incorporation of the iron proto¬ porphyrin IX into the peptide from step C was followed by observing the a- , β- and Soret-band optical spectrum after incremental additions. Spectra were compared to those for the solution spectra of Jis-imidazole porphyrin. E. Purification of Peptide Complex Porphyrin-peptide complex from step D was purified by molecular weight exclusion chromatography using FPLC

(Superdex-75 (Pharmacia-LKB) mobile phase:potassium phosphate buffer; 10-100 mM; pH 6-8) at a flow rate of about 0.5 ml per minute.

Example 2 A. Preparation of Peptide

Peptide and peptide dimer were prepared and purified as described in Example 1.

B. Addition of Iron Protoporphyrin IX Peptide was titrated with a solution of ferroproto- porphyrin IX chloride. Redox titrations indicate the electrochemical asymmetry of the bound iron protoporphyrin IX compounds. The bound iron protoporphyrin IX compounds exhibit respectively distinct redox midpoint potentials of about -220mV and -95mV (versus standard hydrogen electrode (SHE)). Titration of the synthetic peptide complex having only one iron protoporphyrin IX compound bound thereto confirms that the about -220 mV species is the first porphyrin compound complexed by the peptide. The free iron protoporphyrin IX has a redox midpoint potential of less than about -300mV, which differs significantly from the redox midpoint potential of the synthetic peptide complex. Example 3

A. Preparation of Lan muir-Blodαett film

The synthetic peptide complex prepared in Example 1 is deposited as a monolayer film on glass using Langmuir- Blodgett film preparation. Examining the monolayer films indicates that the peptides are in a highly ordered and stable structure with the iron protoporphyrin IX planes oriented at 79±6° with respect to the glass substrate. This indicates that the iron protoporphyrin IX and the supporting α-helical peptides are positioned nearly upright on the glass substrate.

B. Detection device

Current and/or voltage are measured by applying a voltage across electrodes which are comprised of Indium-tin oxide and which hold insulators and the films of the complex prepared in Step A above. The insulators are selected for their transmission of the measured molecule. The density of the synthetic peptide complex is calculated on the basis of its cross-section. For a monolayer film the density is 10¹³ molecules per square centimeter, sufficient for easily assayed electrical signals. The synthetic peptide complexes in the film are substantially completely vectorially asymmetric. In addition, the synthetic peptide complexes may be designed to chemically attach to preferred surfaces.

SEQUENCE LISTING

NUCLEOTIDE AND AMINO ACID SEQUENCE DISCLOSURE PURSUANT TO 37 CFR §1.821(c) .

(1) GENERAL INFORMATION:

(i) APPLICANT: Dan E. Robertson, Ramy S. Farid

William F. DeGrado, and P. Leslie Dutton (ii) TITLE OF INVENTION: Electrically Conducting

Synthetic Peptide Complexes (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Stephen M. Sammut

(B) STREET: Center for Technology Transfer, UNIVERSITY OF PENNSYLVANIA, Suite 300, 3700 Market Street

(C) CITY: Philadelphia

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19104-3147 (V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette-5.25 inch, 1.2 Mb

(B) COMPUTER: AST Bravo IBM PC comp. (386SX)

(C) OPERATING SYSTEM: MS DOS version 3.2

(D) SOFTWARE: WordPerfect 5.1 conv. to ASCII

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Express Mail Label No. TB122054515US

(B) FILING DATE: 12-Jan-1993

(C) CLASSIFICATION: (viii)ATTORNEY/AGENT INFORMATION:

(A) NAME: Cherry, David A.

(B) REGISTRATION NUMBER: 35,099 (C) REFERENCE/DOCKET NUMBER: 19,265 USA

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 923-4466

(B) TELEFAX: (215) 923-2189

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Cys Gly Gly Gly Glu Leu Trp Lys Leu His 1 5 10

Glu Glu Leu Leu Lys Lys Phe Glu Glu Leu

15 20

Leu Lys Leu His Glu Glu Arg Leu Lys Lys

25 30

Leu 31

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Cys Gly Gly Gly Ala Leu Trp Aib Leu His 1 5 10

Leu Ala Leu Leu Aib Leu Phe Ala Leu Leu

15 20

Leu Aib Ala His Leu Ala Arg Leu Aib Leu

25 30

Leu 31

Claims

1. A process for preparing an electrically conducting synthetic peptide complex comprising providing first and second peptides, each of said peptides having a helical structure, and combining said first and second peptides with a plurality of complexing agents, said complexing agents binding with said peptides to provide the synthetic complex with a preselected electrically conductive path.

2. The process according to Claim 1 comprising providing said peptides by solid phase synthesis.

3. The process according to Claim 1 further comprising linking together said peptides to form dimers.

4. The process according to Claim 3 comprising preparing an aqueous solution of said peptides having an amino terminal residue and mixing said solution under conditions wherein said cysteine residues are oxidized to form cystine disulfide bonds.

5. The process according to Claim 3 comprising providing a first peptide having a disulfide bond with a leaving group at the amino terminus and a second peptide substituted with a cysteine residue at the amino terminus, and reacting together said first peptide and said second peptide to form a disulfide linkage.

6. The process according to Claim 3 wherein said dimers are titrated with at least two equivalents of said complexing agent to provide the synthetic peptide complex.

7. An electrically conducting synthetic peptide complex comprising first and second peptides which are spaced in parallel relationship, each of said peptides having a helical structure and being bound by a plurality of complexing agents, said complexing agents having redox potential asymmetry to provide the synthetic complex with a preselected electrical characteristic.

8. The complex according to Claim 7 wherein said peptides comprise α-helices.

9. The complex according to Claim 7 wherein the amino terminal residue of each of said peptides comprises cysteine.

10. The complex according to Claim 9 wherein each of said pairs of said peptides are linked together at said amino termini by a cystine-cystine disulfide bond.

11. The complex according to Claim 7 wherein each of said peptides comprises at least two histidine residues.

12. The complex according to Claim 11 wherein each of said peptides comprises about 6 to about 20 amino acid residues between said histidine residues and a shortest edge to edge distance of at least about 3.6 angstroms.

13. The complex according to Claim 12 wherein each of said peptides comprises about 10 to about 13 amino acid residues between said histidine residues.

14. The complex according to Claim 13 wherein each of said peptides comprises about 13 amino acid residues between said histidine residues.

15. The complex according to Claim 11 wherein each of said peptides comprises at least one neutral residue between said histidine residues.

16. The complex according to Claim 15 wherein said neutral residue is selected from the group consisting of phenylalanine, alanine, glycine, isoleucine, leucine, tyrosine, serine, valine, threonine and methionine.

17. The complex according to Claim 16 wherein said neutral residue is selected from the group consisting of phenylalanine, alanine, glycine, isoleucine, leucine and valine.

18. The complex according to Claim 17 wherein said neutral residue is phenylalanine.

19. The complex according to Claim 11 wherein eac of said peptides comprises at least one arginine residue which is located within about four amino acid residues from the carboxy terminus of each of said peptides.

20. The complex according to Claim 19 wherein eac of said peptides comprises at least one arginine residue which is located within about two to about five amino acid residues from said histidine residue.

21. The complex according to Claim 11 wherein each of said peptides comprises at least one tryptophan residue.

22. The complex according to Claim 11 wherein the amino terminus of each of said peptides comprises the amino acid sequence Cys-Gly-Gly-Gly-.

23. The complex according to Claim 7 wherein said complexing agent comprises a compound which is capable of forming complexes with heteroatom-containing compounds.

24. The complex according to Claim 23 wherein said heteroatom-containing compounds comprise histidine.

25. The complex according to Claim 24 wherein said complexing agent comprises a derivative of porphine or an organometallic complex which comprises a metal selected from the group consisting of ruthenium, cobalt, nickel and copper and ligands selected from the group consisting of pyridine, pyrrole, thiophene, furan and 2 , 2'-bipyridine.

26. The complex according to Claim 25 wherein said organometallic complexing agent comprises a substituted or unsubstituted porphyrin, said substituents comprising alkyl, aryl, alkenyl, hydroxy, alkylcarboxy or halo.

27. The complex according to Claim 26 wherein said porphyrin comprises a central metal selected from the group consisting of iron, zinc, magnesium, copper and nickel.

28. The complex according to Claim 27 wherein said organometallic complexing agent comprises chlorophylls, he e, octaethylporphyrin or meso-tetraphenylporphyrin.

29. The complex according to Claim 28 wherein said organometallic complexing agent comprises iron protoporphyrin IX.

30. An electrically conducting synthetic peptide complex wherein said complex comprises a pair of peptides having the following amino acid sequence: Cys-Gly-Gly-Gly- Glu-Leu-Trp-Lys-Leu-His-Glu-Glu-Leu-Leu-Lys-Lys-Phe-Glu-Glu- Leu-Leu-Lys-Leu-His-Glu-Glu-Arg-Leu-Lys-Lys-Leu, wherein said peptides are linked together at residue 1 (cysteine) and wherein said peptide complex comprises two iron protopor¬ phyrin IX, wherein a first of said iron protoporphyrin IX compounds is complexed between the residues 10 (histidine) and a second of said iron protoporphyrin IX compounds complexed between the residues 24 (histidine) .

31. An electrically conducting synthetic peptide complex comprising the following formula: R-C(=0)-CH(NH₂)-CH₂-S-S-CH₂-CH(NH₂)-C(=0)-R₁ I wherein R and R_! are each independently a peptide having the following amino acid sequence: -Gly-Gly-Gly-Glu-Leu-Trp-Lys-

* Leu-His-Glu-Glu-Leu-Leu-Lys-Lys-Phe-Glu-Glu-Leu-Leu-Lys-Leu- #

His-Glu-Glu-Arg-Leu-Lys-Lys-Leu, and wherein said peptide complex comprises two iron protoporphyrin IX compounds, wherein a first of said iron protoporphyrin IX compounds is

* complexed between the His residues of each of R and R_ and a second of said iron protoporphyrin IX compounds complexed

# between the His residues of R and R,.

32. An electrically conducting synthetic peptide complex comprising first and second peptides, said peptides being helical and spaced in parallel relationship, and two organometallic complexing agents which are selectively bound to said peptides and have a shortest edge to edge distance of about 3.6-35 angstroms wherein said complexing agents are located at selective sites in the complex and impart an electrical characteristic to the synthetic peptide complex.