DESCRIPTION

LARGE SYNTHETIC COMPOUNDS BASED ON ELECTRON DONOR AND ELECTRON ACCEPTOR INTERACTIONS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods and compositions useful in the production of large molecular systems with predetermined structure, properties and function. In particular, these structures are derived from the folding of synthetic molecular chains composed of at least some abiotic units. These structures can further be modified to intercalate into DNA.

2. Description of Related Art

In recent years, the chemistry of large synthetic molecules that fold into well- defined structures has been an area of great interest (Lindsey, 1991 ; Amabilino et ai, 1995; Whitesides et al., 1991 ; Lehn, 1990; Chambron et ai, 1994; Ashton et al, 1991 ; LaBrcnz & Kelly, 1995). Much of this interest stems from the recognition that these molecules can be expected to have some of the important properties found in biological macromolecules, yet also may contain some unique chemical properties not found in naturally occurring macromolecules.

Large biological molecules have complex, yet stable structures derived in large part from numerous intramolecular non-covalent interactions. The secondary structure of these large molecules enable them to have the tremendous range of function required to carry out the processes required for life itself. Clearly, the construction of molecules that fold or assemble predictably into large, well-defined structures would represent an important advance towards the design of molecules on a size scale similar to that of biological macromolecules.

Biomimetic compounds are defined generally as molecules that have the general shape of a biological molecule, such as DNA, peptides or proteins, with few if any of the naturally occurring biological building blocks in their structure. A particular class of biomimetic compounds, known as peptidomimetic compounds, are those compounds which have the general folding properties of peptides or proteins with very few if any naturally occurring amino acids in their structure. These peptidomimetic compounds can exist as independently folded structures or can be placed within an existing protein to effect or enhance a specific function.

Another goal in designing large synthetic molecules is the development of molecules which interact with biological macromolecules, such as DNA, to perform a specific function. Numerous molecules, including therapeutic agents employed clinically, intercalate into DNA. Intercalation can be defined as insertion of a planar group between the base pairs of a DNA double helix. This interaction long has been appreciated as an important mechanism for the development of compounds with therapeutic anti-cancer (anti-tumor) activity, and further for the development of compounds with sequence specificity towards the DNA target (Foye).

While most compounds studied that exhibit intercalalive binding are molecules with a single intercalating moiety (Lerman, 1961 ; Wilson & Jones, 1982), polyintercalators have been shown to have an increased affinity for the DNA, and the larger number of base pairs contacted can provide increased activity such as sequence specificity. While the limit to the number of intercalating groups that can be positioned along the backbone is not known, efforts to extend the interaction beyond bis- intercalation have been largely unsuccessful due to the synthetic methods employed and the traditional design of intercalating compounds. Despite the broad range of intercalating agents presently known, there remains a need for the development of polyintercalating agents with binding beyond b/.v-intercalation and, preferably, with defined sequence specificity for use in therapeutic applications.

It is well appreciated that drugs in general, and anticancer drugs in particular, are discovered using a wide variety of approaches, including experimental screening, rational drug design and fortuitous detection. A compound that would allow for rapid yet precise manipulation of side chains for rational design, for example, being amenable to random placement using synthetic methods such as combinatorial chemistry, would provide a significant advance in the discovery process for new intercalating agents for therapeutic use.

At present, while tools are available through which large molecular structures may be constructed, there remains a need for further development of these systems in order to decouple the folding properties of the molecule from the molecular properties and function of the molecule. This will allow for the rational systematic design of large molecules with predetermined folding properties and functions. There also remains a need for the development of synthetic systems designed to interact, in a predetermined manner, with existing biological molecules.

SUMMARY OF THE INVENTION

It is, therefore, a goal of the present invention to provide compositions and methods relating to the construction of synthetic molecules that fold or assemble into large well-defined structures, biomimetic compounds, or interact with existing biological molecules. Compounds arc provided comprising at least two alternating units of electron donor and electron acceptor stacking units, said units sequentially linked by a linking unit where the units assemble into a stacked structure. Also provided are compounds of at least two alternating units of electron acceptor stacking units, where the units are sequentially linked by a linking unit to form a structure that intercalates into DNA.

In one embodiment, the compounds of the present invention can be described by the following formula:

-[-Bι-(CH ₂ ) _n -X _m -Y _o -X _m -(CH ₂ ) _p -B ₂ — ]— [A] wherein individually at each occurrence, each of B| or B ₂ is an electron donor or electron acceptor monomer group, with the preferred groups selected from Tables 1-3, wherein individually at each occurrence, each X is a linking unit group selected from amino acid, derivatives of amino acids, nucleic acid, nucleic acid derivatives, saccharide, polysaccharide, amide, ester, ether, phosphate diester, amine, imine, thioether, ketone, carbonate, carbamate, urea, guanidinium, thiourea, thioester, sulfone, sulfoxide, alkyl, alkenyl, alkynyl, glycol, polyglycol, wherein independently at each occurrence each X can be substituted with R selected from alkyl, alkenyl, alkynyl, hydroxyl, aryl, thiol, amine, carboxy, amide urea, thiourea, carbonate, carbamate, ether, thioether, nitro, ketone, aldehyde, sulfone, sulfoxide, thioether, heterocycle, carbohydrate, nucleic acid, amino acid, phosphate, phosphonium, chelating agents, biological targeting agents or antimetabolic agents, wherein n is 0-10, wherein p is 0-10, wherein m is 0-20, wherein the total number of carbons in X is less than or equal to 100, preferably less than 50, most preferably less than 30 and the total number of carbons in R is less than or equal to 30.

In particular embodiments, Y can be substituted with R, wherein R is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a hydroxyl group, an aryl group, a cycloalkyl group, a thiol, an amine, a carboxy group, an amide urea, a thiourea, a carbonate group, a carbamate group, an ether, a thioether, a nitro group, a ketone, an aldehyde, a sulfone, a sulfoxide, a thioether, a heterocycle, a carbohydrate, a nucleic acid, an amino acid, a poly amino alkyl group, a phosphate, a phosphonium group, a chelating agent, a biological targeting agent, a DNA damaging agent, a cleaving agent and an antimetabolic agent; and each X is a linking unit group selected from the compounds listed above for linking unit groups. Likewise B| is an

electron donor or electron acceptor monomer group, with the preferred groups selected from Tables 1-3.

While conventional methods using primarily biologically based building blocks may be sufficient in certain situations, e.g.,. where the functional groups do not interfere with the control of the higher order structure determined by the primary sequence, it will not suffice where the introduction of the functional groups interrupts the folding pattern of the large molecular system. In such a case, it will be desirable to use a system where the function of the molecule is de-coupled from the folding properties of the compound as a whole. The present invention provides a solution to these needs by providing large molecular structures where the higher order structure of the complex is controlled by interactions separate and distinct from the functional groups appended. The present invention provides compounds that fold into a defined secondary structure based on the non-covalent interactions between alternating electron-rich donor groups and electron- deficient acceptor groups linked sequentially via a linking unit as shown in FIG. 1.

A range of compounds with a wide variety of electron donor monomers, electron acceptor monomers and linking units, fall within the scope of the present invention. In one embodiment of the invention, Bi is an electron acceptor moiety and B ₂ is an electron donor moiety, sequentially linked by a linking unit to form an alternating array which assembles into a stacked secondary structure. Preferred embodiments of this aspect of the invention include electron donor and electron acceptor units provided in Tables 1-3, with compounds which are planar and aromatic being most preferred. In particular embodiments of the present invention wherein Bj is selected from the group consisting of 1 ,3.5-Benzenetriamine (TAB); 1,4-Diphenylbutadiene; 1 ,5-dialkoxynaphthalene; 1-

Methylnaphthalene; 1-Naphthol; 1-Naphthylamine; 13,14-

Dithiatricyclo[8.2.1.0 ^4J ]tetradeca-4,6, 10, 12-tctraene (DDDT); 2,4,6-

Tris(dimethylamino)-l,3,5-triazine (TDT); 2-Methylnaphthalene; 2-Naphthol; 2- Naphthylamine; Acenaphtene; Anthracene; Benzene; Bromodurene; Dibenzo[cv/]phenothiazine; Durene; Durenediamine (DAD); Fluoranlhene; Fluorene;

Hexamethylbcnzene (HMB); Indenc; indole and substituted indoles;

N, N, N',N'-Tetramethyl-/7-phenylenediamine (TMPD); N,N-Dibenzyl-m-toluidine; N,N- Dimethylaniline (DMA); Naphthalene and substituted naphthalenes; Nucleic acid bases; />-Phenylenediamine (PD); Pentamethylbenzene; Phenanthrene;

PyreneTetrakis(dimethylamino)ethylene (TDAE); Tetralin; Tetrathiotetracene; Triethylarnmonium (TEA); and Triphenylmethanol.

In other embodiments, B ₂ is selected from the group consisting of 1,2,4,5- Tetracyanobenzene (TCNB); 1,3,5-Tricyanobenzene; 1 ,3,5-Trinitrobenzene (TNB); 1,3,7,9-Tetramethyluric acid (TMU); 1,4-Dicyanobenzene; 1.4-Naphthoquinone; l l,l l,12,12-Tetracyanonaphtho-2,6-quinodimethane (TNAP); 2,2-Diphenyl-l- picrylhydrazyl (DPPH); 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ); 2,3-Dichloro-/?- benzoquinone; 2,3-Dicyano-p-benzoquinone; 2,4,5,7-Tetranitro-9-fluorenone (TENF); 2,4,6-Trinitrotoluene (TNT); 2,4,6-Trinitroxylene; 2,4,7-Tetranitro-9- fluorenylidenemalononitrile; 2,4,7-Trinitro-9-fluorenone (TNF); 2,4-Dinitro-9- fluorenone; 2,5-Bis(rnethylamino)-p-benzoquinone (BAQ); 2,5-Diethoxy-p- benzoquinone (DEQ); 2,6-Dibromo-/?-benzoquinone; 2,6-Dinitro-p-benzoquinone; 3,4- Benzopyrene (BP); 7,7,8, 8-Tetracyanoquinodimethane (TCNQ); 9, 10- Anthraquinone; 9- Fluorenylidenemalononitrile; Benzoquinone; Benzotrifuroxan (BTF);

Dibromopyromellitic dianyhydride; Dinitrobenzene; Dinitronaniline; Dinitrophenol; Duroquinone; Hexacyanobutadiene (HCBD); m-Dinitrobenzene; Maleic anhydride;

Mellitic trianhydride; Nitrobenzene; o-Dinotrobenzene; /?-Benzoquinone; p- Dinitrobenzene; Phenanthroquinone; Phthalic anhydride; Prioric Acid; Proflavin Pyromellitic dianhydride (PMDA); Tetrabromo-ø-benzoquinone (o-bromanil) Tetrabromo-p-benzoquinone (bromanil); Tetrachloro-o-benzoquinone (c-chloranil) Tetrachloro-p-benzoquinone (chloranil); Tetrachlorophthalic anhydride (TCPA)

Tetracyano-/?-benzoquinone; Tetracyanoethylcne (TCNE); and Tetraiodo-/?- benzoquinone (iodanil).

In a specific embodiment, the electron donor moiety is 1 ,5-dialkyloxynapthalene and the electron acceptor moiety is 1,4,5,8-napthalenetetracarboxylic diimide, to provide

a compound of the formula shown below In a furthet prefened embodiment, the linking unit is an amino acid, with aspaitic acid being most preferred

In preferred embodiments the compound has the repeating stiucture

[BJ

In a moie defined embodiment, X is aspaitic acid, n is 2, m is 1 and p is 3

Anothei aspect ot the piesent invention provides a compound comprising a plurality of electron acceptoi stacking units, wherein the units die linked b> a linking unit In piefeπed embodiments the compound has the general iormula

-[-B,-(CH ₂ ) _n -X _π -(CH ₂ ) _p -B _| -l-

wherein

Bi is an electron acceptor stacking unit,

-(CH ₂ ),,-X _m -(CII ₂ ) _P - is a linking unit and X is a linking unit gioup, n is 0- 10 m is 0-20 and p is 0-10 with the provision that the sum of n, m and p must be equal to oi gicatei than 1

The linking unit gioup is selected from the group consisting ot an amino acid, an amino acid derivative, a nucleic acid, nucleic acid deuvatives, a sacchaπde, a

polysaccharide. an amide, an ester, an ether, a phosphate diester, an aminc, an imine, a thioether, a ketone. a carbonate, a carbamate, a urea, a guanidinium group, a thiourea, a thioester, a sulfone, a sulfoxide, an alkyl group, an alkenyl group, an alkynyl group, a glycol, a polyglycol. In further embodiments, independently, each X can be substituted with R, wherein R is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a hydroxyl group, an aryl group, a thiol. an amine. a carboxy group, an amide urea, a thiourea, a carbonate group, a carbamate group, an ether, a thioether, a nitro group, a ketone, an aldehyde, a sulfone, a sulfoxide, a thioether, a heterocycle, a carbohydrate, a nucleic acid, an amino acid, a phosphate, a phosphonium group, a chelating agent, a biological targeting agent, a DNA damaging agent, a cleaving agent and an antimetabolic agent.

In another preferred embodiment the compound has the general formula:

H-B,- ⁽ CrL ⁾ _n -X _m -Y _υ --X _m - ⁽ CH ₂ ⁾ _p -BH-

[I>J

wherein

B | is an electron acceptor stacking unit; -(CH ₂ ) _n -X _n -(CH ₂ ) _r - is a linking unit and X is a linking unit group; n is 0- 10; m is 0-20; o is 0-20; and p is 0- 10 with the provision that the sum of n, m o, and p must be equal to or greater than

1. In more defined terms. Y can be substituted with R, wherein R is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a hydroxyl group, an aryl group, a cycloalkyl group, a thiol. an amine. a carboxy group, an amide urea, a thiourea, a carbonate group, a carbamate group, an ether, a thioether, a nitro group, a ketone. an aldehyde, a sulfone. a sulfoxide, a thioether, a heterocycle. a carbohydrate, a nucleic acid, an amino acid, a poly amino alkyl group, a phosphate, a phosphonium

group, a chelating agent, a biological targeting agent, a DNA damaging agent, a cleaving agent and an antimetabolic agent, and each X is a linking unit group selected from the compounds listed above for linking unit groups

In prefened embodiments, the biological targeting agent may be selected from the group consisting of a cytokine, a growth factor, a steroid hoimone, an antibiotic and an ohgonucleotide In othei embodiments, the anlimetabolic agent is selected from the group consisting of AraC, deoxyuπdinc, dideoxyundine. fluorouiacil. thioguamne, meicaptopuπne, azaπbine, AZT and LOMPD In yet tuither embodiments the DNA damaging agent is selected from the group consisting oi encdiyne. a biomo acid, a nitrosouiea, an ethylenimine, an alkylsulfonate. an amide, an epoxidc. daunoiubiαn, pipeiazinedione and mitomycin C

In paiticulai embodiments, the election acceptoi unit is selected tiom the group consisting of 1.4,5.8-Naphthalene tetiacai boxvhc acid diimide 7-Amιnoactmomycιn D,

Acπdine, Actinomycin D, Adnamycin, Amsacπnc. Bisantiene, Daunomycin, Daunorubicin, Dnmidium, Ethidium bromide Methyl violagen Mitioxantrone, Naphthalene and substituted naphthalenes, Phenylquinolone-H-caiboxamides. Porphyπn analogs, Propidium iodide, Quinacnnc; Quinolone and Sanguinanne In a moie particular embodiment, the election acceptoi unit is of 1 ,4,5,8-Naphthalcne tetracaiboxyhc acid diimide

The present invention also relates to the constiuction ot huge molecules designed to interact with biological molecules such as DNA intcicalaioi s Moic specifically, the piesent invention provides polyinteicalatois that can be extended beyond bι \- inteicalation to inciease affinity and sequence specif icity toi taiget DNA

In one embodiment of the piesent invention, polyacceptoi compounds aic provided compπsing election deficient monomei units linked in scnes via a linking unit. Piefened electron deficient monomei units aic planai and aromatic to allow foi intercalation into DNA, with the election deficient monomei units ol Table 3 being most

preferred. In a further pieferred embodiment, the electron-deficient monomer units are aromatic diimide groups, with being most preferred as shown below In a specific preferred embodiment, a compound for intercalation is provided where the electron deficient monomers are 1 , 4, 5, 8-napthalenetelracarboxyhc diimide and the linking unit is a peptide, in particular, gly-gly-gly-lys.

In another embodiment ol the present invention, poly-donor compounds aie provided compπsing electron nch monomer units linked in series via a linking unit In a preferred embodiment, the election-rich monomer units aie selected from the groups listed in Table 1 with 1 ,5-dιalkoxynapthalene groups being most piefened.. In a iuithei preferred embodiment, a compound lor interaction with a polyacceptoi compound is provided where the electron nch monomers aie selected to interact with the pieferred electron def icient monomers of the poly acceptoi These interactions aic well characterized in the art as exemplified in Hei bstein ( 197 1 ) incorporated herein by reference.

The synthetic methods used to prepare the compounds ol the present invention aie essentially those used for standard peptide synthesis, with solid phase methods being particularly preferred Specific examples for the synthesis of compounds of the present invention aie provided in Examples 1 and 3.

A pai ticular advantage ot the compounds of the piesent invention is the opportunity foi combinatonal synthesis. Specifically, the solid phase synthesis of these compounds allows for combinatorial methods to be used for the production of a wide

- I I - variety of compounds to be produced. Specific examples of combinatorial techniques as applied to the present invention are provided in Examples 6 and 7.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 shows a general schematic representation of a secondary structural element based on electron donor and electron acceptor interactions.

FIG. 2A and FIG. 21$ FIG. 2A and FIG. 2B show electron acceptor (FIG. 2A) and electron donor (FIG. 2B) monomer units. Compounds 1. 2, 3 and 4 were used to prepare a co-crystal for studies involving linker unit optimization.

FIG. 3A, FIG. 3B and FIG. 3C. FIG. 3A and FIG. 3B show x-ray structure of the 3-4 co-crystal. FIG. 3A shows a view down the h axis showing the π-stacking of the two molecules 3 and 4. Only the major component of the disordered side-chain is shown. 4 is shown as dashed lines, while 3 is shown as thermal ellipsoids drawn to the 30% probability level. The molecules each lie around crystallographic inversion centers separated by a Vt b-axis translation (3.46 A). FIG. 3B shows a portion of the extended π- stacking arrangement of 3 and 4 along the b axis. Atoms arc represented as spheres of an arbitrary size. The solvent molecules have been omitted for clarity. FIG. 3C shows a computer-generated representation of 16 (Scheme 4) based on X-ray structure of the 3-4 co-crystal and calculated structure of the backbone.

FIG. 4A and FIG. 4B. FIG. 4A and FIG. 4B show two possible modes of intercalation FIG. 4 A shows a mode of intercalation in which the aromatic groups aie stitched through the DNA double helix such that theii hnkei segments reside in the major and minor grooves in an alternating fashion. FIG. 4B shows intercalation into the DNA double helix where the linker segments reside entirely within the major groove.

FIG. 5. FIG. 5 shows viscometπc titrations of 1 (Δ); 2 (♦ ); 3 (■); and 4 (• ) with sonicated calf thymus DNA, carried out in TE bufler ( 10 mM Tris. 1 mM EDTA) with 50 mM NaCl at 20°C Since the monomer was only partially bound at the concentrations used in the viscosity experiment, the [compound]/[DNA base pairs j values weie corrected based on the known association constant (K _a = 1 0 x l O^ M ' ) obtained liom the spectrophotometπc titration of 1 with calf thymus DNA

FIG. 6. FIG. 6 shows unwinding of closed circular supercoiled plasmid DN A by compounds 2 (♦ ); 3 (■), and 4 (• ), conducted in a Cannon-Ubbelhode scmi-imci o dilution viscometer (size 75) in TE bufler with 50 mM NaCl according the method of Revet et al. ( 1971 ) Plasmid pMC1403 was prepaicd essentially according to Sambrook et al ( 1989), and purified using a CsCl gradient ulti acentπf ugation in the presence of propidium iodide Viscosity was measuied at DNA concenti ations of ~2 x 10 ^ M. For compounds 2-4, the compound/DNA base pan ratio at the at the unwinding equivalence point was independent of DNA concentration, indicating that, under these conditions, the compounds were fully bound. For monomer 1, equivalence point iatios at three different DNA concentrations weie plotted vs the concentration of DNA and the true equivalence point was extrapolated accoiding the Revet et al ( 197 1 ). Unwinding angles were calculated based on an unwindin *&g angle for ethidium bromide of 26°

FIG. 7. DNAse I tootpπnting analysis of 78-meι DNA with the "+" strand labeled on its 5 ^' end, in the presence o( compounds 22-25. Lanes labeled AG and TC represent the purine- and p\ πmιdιne-spccιfιc sequencing leactions Lane CO contains DNA without DNAse I. Lane C contains DNA with DNAse I but no compound Foi compound 22, lanes 1-5 contain 0 5, 5, 50, 100 and 500 μ M compound, respectively.

n

For compounds 23-25, lanes 1 -5 contain 62 5, 125. 250, 500, and 1000 nM compound respectively

FIG. 8. DNAse I footpπnting analysis of 78-meι DNA with the "-"' strand labeled on its 5 ^' end, in the presence of compounds 22-25 Lanes labeled AD and TC icpiesent the punne- and pyπmidine-specific sequencing icactions Lane CO contains DNA without DNAse I Lane C contains DNA with DNAse I but no compound Foi compound 22. lanes 1 -5 contain 0 5 5. 50. 100 and 500 μ M compound, lespectivcly Foi compounds 23-25. lanes 1 -5 contain 62 5, 125, 250 500 and 1000 nM compound, lespectively

FIG. 9A and FIG. 9B. Schematic icpiesentation of the binding of tetrainteicalatoi 25 to double stranded DNA by FIG 9A w eav ing into and out of the double helix such that the hnkei s leside in both groov es oi by FIG 9B mteicalation solely from one side ot the double helix

UG. 10. Simplified mechanistic scheme loi the dissociation oi intcicalators 23- 25 in which the dimei 23 binds coopeiatively in pan s All thiee compounds dissociate from DNA in a similar step-wise pattern, in which the of I -i ate s depend on the occupancy of neighbonng intercalative sites

FIG. 11. tπmei

FIG. 12. dimei

FIG. 13A, FIG. 13B and FIG. 13C UV dissociation profiles (λ=384 nm) of compounds 23, 24 and 25 from poly(dAdT) in 2'/o S DS (giey) and the calculated theoietical curves (black), according to a model in which the dimei 23 binds coopei atively (FIG 10- 12) The icsulting l ate constants f oi the elemental \ steps of dissociation aie civ en m Table 7

FIG. 14. Absorbance spectrum of tetramer 25 ( 1. 1 6 x 10 \M. 50 mM TRIS buffer, pH 7.0) in the absence (solid line) and presence (dashed line) of excess calf thymus (CT) DNA.

FIG. 15. Binding isotherm of 22 and calf thymus DNA. determined spectrophotometrically using the change in absorbance of 22 at 386 nm upon addition of DNA. The data was fit to the McGhee-von Hippel equation (McGhee and von Hippel, 1974), where r = the concentration of bound ligand divided by the total concentration of DNA, and c = the concentration of free ligand.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to compositions and methods for the construction of large molecular structures that fold or assemble into a w ell defined higher order structure. Modification of these structures permits the construction of compounds which interact with biological molecules in a specific mannei such as DNA intercalation. The principal underlying this technology is that by using abiotic monomer units that will define the folding pattein oϊ the higher order structure, regardless of the f unctional groups attached to the linking unit, it is possible to construct stably-folded, functional, abiotic large molecular structures. A variation on this technology allows for the construction of molecules which interact with a biological molecule in a specified manner, such as DNA intercalation, independent from the functional groups introduced into the linking unit. While a DNA intercalating molecule is an example of a polyacceptor complex, the present invention also allows for the construction ol polydonoi complexes which can interact with polyacceptor compounds to form large molecular arrays.

This decoupling of structure, molecular properties and lunction constitutes a significant advance ovei using biologically-based monomers such as amino acids or nucleic acids. By using abiotic monomer units to def ine the folding properties of the

compounds, there is no constraint to the biological folding patterns such that would be required with biologically based monomer units

A. Building Blocks

Electron donor monomei units of the present invention are compounds with a low ionization potential. The general structure of election donoi compounds contemplated by the present invention include aromatic hydrocarbons and heterocycles such as benzene and particularly napthalcne and napthalene derivatives of which about ninety different types are known, metallocenes, porphyπns and cooidination complexes such as the metal oxinates of 8-qιunolιnol Furthei, representative examples of monomei units useful as electron donating units foi the present invention aie provided in Table 1 with planai aromatic groups preferred and with 1 ,5-dιalkoxynapthalene being most preferred as shown in FIG. 2B.

TABLE 1 -Electron Rich Monomer Units

1 ,3,5-Benzeπetπamιne (TAB )

1 ,4-Dιphenylbutadιene 1 ,5-dialkoxynaphthalene

1 -Methy Inaphthalene

1 -Naphthol

1 -Naphthylamιne

13.14-Dιthιatπcyclo[8.2.1.0 ^{t 7} Jtetιadeca-4.6, lϋ, 12-tetraene (DDDT) 2,4,6-Tπs(dιmethylamιno)- l ,3,5-tπazιne (TDT)

2-Methy Inaphthalene

2-Naphthol

2-Naphthylamιne

Acenaphtene Anthracene

Benzene

Bromodurene

Dιben/o[c,c/lphenothιazιne

Durcnc

Durenediamine (DAD) Fluoranthcne

Fluorene

Hexamethylbenzene (HMB)

Indene

Indole and substituted indoles N, N, Λ^',N'-Tetramethyl-/j-phenylenedιamιne (TMPD)

N,N-Dιbenzyl-m-toluιdιne

N,N-Dιmethylanιlιne (DMA)

Naphthalene and substituted naphthalenes

Nucleic acids bases p-Phenylenediamine (PD)

Pcntamcthylbenzene

Phenanthrene

Pyrene

^' I etrakιs(dιmethylamιno)ethylene (TDAE) Tetralin

Tetrathiotetiacene

Tπethylammonium (TEA)

Tπphenylmethanol

Election acceptoi monomei units of the piesent invention aie compounds which have a high election af f inity Electron acceptoi s may be classif ied accoi ding to gencial stiuctuie type and substituents The gencial structure of election acceptoi units contemplated by the present invention include olef inic compounds such as tetracyanoethylene and hexacyanobutadiene, substituted aiomatic compounds such as substituted benzenes and polynitionapthalencs and substituted ai omatic ketones and

quinones such as substituted benzoquinones, napthaquinones, fiourenones and anthiaquinones (Herbstein, 1971 )

Fuithei , icpresentative examples of election acceptois contemplated by the present invention are provided in Tables 2 and 3, with planai aiomatic groups preferred, and 1,4,5,8-napthalene-tetιacarboxylιc acid diimide being most piefeiied, as shown in FIG 2A

TABLE 2-Electron Deficient Monomer Units

1 , 2,4,5-1 etracyanobenzene (TCNB)

1 ,3,5-Tι icyanobenzene l ,3,5- ^' J πnιtrobenzne (TNB)

1 ,3,7,9-Tetramethyluπc acid (TMU)

1 ,4-Dιcyanoben/ene 1 ,4-Naphthoquιnone

1 1, 1 l , 12, 12-letιac> anonaphtho-2,6-quιnodιnιethane (I N API

2,2-Dιphenyl- 1 -pici v Ihydi a/yl (DPPH )

2,3-Dιchloιo-5,6-dιcvanoben7oquιnone (DDQ)

2,3-Dιchloιo-/;-ben7oquιnone 2,3-Dιcyano-/?-ben/oqumone

2,4,5,7-Tetιanιtro-9-fluoιenone (TENF)

2,4,6-'Innιtιotoluene (TNT)

2,4,6-Tι mitroxylene

2,4,7-Tetranιtro-9-πuorenyhdenemalononιtπle 2,4,7-Tnnιtιo-9-fluoιenone (TNF)

2,4-Dιnιtιo-9-fluoιenone

2,5-Bιs(methylammo)-/;-benzoquιnone (BAQ)

2,5-Dιethoxy-/;-benzoquιnone (DEQ)

2,6-Dιbromo-/?-ben/oquιnone 2,6-Dinitio-/?-benzoquinone

3,4-Ben/opyrene (BP)

7,7,8, 8-Tetracyanoquιnodιmethanc (TCNQ)

9, 10-Anthraquinone

9-Fluorenyhdenemalononιtπle

Benzoquinone Benzotπturoxan (BTF)

Dibromopyromelhtic dianyhydπde

Dinitiobenzene

Dinitionanihnc

Dinitiophenol Duroquinone

Hexacyanobutadiene (IICBD)

/7!-Dιnιtrobenzene

Maleic anhydride

Melhtic tnanhydπde Nitrobenzene

(V-Dinotrobenzenc

/7-Benzoquιnone p-Dιnιtrobenzene

Phcnanthroquinone Phthahc anhydride

Pπoπc Acid

Proflavin

Pyromelhtic dianhydπdc (PMDA)

Tctrabromo-<?-ben/oqιunone (r;-bιomanιl) Tetrabtomo-p-benzoquinone (bromanil)

Tetiachloi o-o-benzoquinone (o-chloraiπl)

Tetrachloro-/7-benzoquιnone (chloraml)

Tetrachloiophthahc anhydπdc (TCPA )

Tetracyano-/?-benzoquιnone Tetracyanoethylene (TCNE)

Tctraιodo-/?-benzoquιnone (lodanil)

TABLE 3-Electron Deficient Monomer Units Preferred for DNA Intercalation

1.4,5,8-Naphthalene tetracarboxyhc acid diimide

7-Amιnoactιnomycιn D Acπdine

Actinomycin D

Adπamycin

Amsacπne

Bisantrene Daunomycin

Daunoiubicin

Diimidium

Ethidium bromide

Methyl violagen Mitioxantrone

Naphthalene and substituted naphthalenes

Phenylquιnolone-8-carboxamιdes

Porphyπn analogs

Propidium iodide Quinacπne

Quinolone

Sangu marine

Linking unit groups of the present invention aie compounds which contain bifunctional groups for appending between monomer units Representative examples ot linking units contemplated by the present invention aie composed of ammo acids, including amino acids with simple aliphatic side chains (e g. glycine. alanine, valine, leucine and isoleucine), ammo acids with aiomatic side chains (e g phenylalanine, tryptophan, tyrosine, and histidine), amino acids with oxygen and sulfui containing side chains (e.g. serine, threonine, methionine and cysteine). amino acids with side chains containing caiboxylic oi amide groups (e g. aspartic acid, glutamic acid, asparagine and

glutamine), and amino acids with side chains containing strongly basic groups (e g lysine and aiginine) and pioline Deuvatives of the above desci ibed amino acids aie also contemplated as linkei units An amino acid dei ivative as used herein is any compound that contains within its structuie the basic amino acid coie of an α amino- substituted caiboxylic acid with representative examples including azaseπne, fluoroalanine, GABA, ormthine, norleucine and cycloseπne Peptides derived from the above desciibed amino acids can also be used as monomei units Repiesentative examples include both naturally occurring and synthetic peptides f rom 1 -about 20 amino acids in length, with peptides from 1 5 amino acids pi efei ied

The linking units according to the piesent invention also may be composed oi nucleobase compounds As used heiein the teim nucleobase letet s to any moiety that includes within its structuie a puπnc, a pyπmidine, a nucleic acid nucleosidc, nucleotide oi dei ivative of any ol these such as a protected nucleobase put inc analog, pyrimidine analog oi fohnic acid analog

As shown in Formula A, linking units of the piesent invention may also include moieties X as previously defined, each of which may contain a substituent R as def ined picviously

-( C I I ) , - \ , -K H ) , B -

Repiesentative examples of alkanes include methane ethane stiaight chain, blanched oi cyclic isomers of propane, butane pcntane, hexane heptane, octane nonane and dccane Repiesentative examples ol alkenes include methene ethene straight chain branched oi cyclic isomei s of propene butene pentene hexcne, heplene υctene nonene and decene Repiesentative examples of alkynes include methyne ethyne, stiaight chain blanched oi cyclic isomei s of pi opyne butyne pentyne, hex\ ne heptyne octyne nonyne and decyne Wheie substitution of ΛIΛ alkyl group with a halide is contemplated, lepiesentative examples of halides include chloπne bromine fluonne and iodine

Repiesentative examples of hv dioxyalkyls include alcohols of methane ethane straight

chain, branched or cyclic isomers of propane, butane, pentane. hexane. heptane, octane, nonane and decane. Representative examples of glycols contemplated mclude diols of methane, ethane, straight chain, branched or cyclic isomers of propane, butane, pentane. hexane, heptane, octane, nonane and decane. Representative examples of polyglycols include polyethylene glycol, polypropylene glycol and polybutylene glycol as well as polyalkclene glycols containing combinations ol ethylene. propylene and butylene. Representative thiols include alkyl thiols of ethane, stiaight chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane. Representative examples of sacchaπdes include ribose. arabinose. xylose glucose, galactose and othei sugar derivatives composed of chains from 2-7 carbons

Representative polysaccharides include combinations of the saccharide units listed above linked via a glycosidic bond Representative chelating agents mclude EDTA, EGTA, DTPA, DOTA, ethylene diamine, bipyradinc, 1 , 10-phenanthrohne, crown ether, aza crown, gly-gly-his tπpeptide and catechols Repiesentativ e nucleotide. nucleoside antimetabolites include Ai aC. deoxyundine, dideoxyuπdine. I luoiouracil. thioguanine. mercaptopunne, azaπbine, AZT and LOMPD Repiesentative cleaving agents include Fe-EDTA, Cu-Gly-Gly-His, Fe-bleomycin and Cu- 1 , 10-phenanthrohne. Repiesentative DNA damaging agents include alkylating agents such as enediyne, biomo acids, nitrosoureas, ethylenimines, alkylsulfonates, amides epoxides. daunorubicin, piperazinedione and mitomycin C. Repiesentative biological tai geting agents include cytokines, growth factois. steroid hormones such as estrogen, pi ogestins and androgens, antibiotics, compounds such as ncocar/inostatin which hav e a piefeience foi certain types of DNA sequences and oligonucleotides to selectively bind substantially complimentary sequences, As used herein, a substantially complementary sequence is one in which the nucleotides generally base pan with the complementary nucleotide and in which there aie very few base pair mismatches

B. AEDAMers (Alternating Electron Donor-Acceptor Units)

Currently, laige synthetic molecular structuies aie constructed using primarily biological buildin blocks, such as amino acids oi nucleic acids A mote treneral

approach would be to use "abiotic elements". Such abiotic building blocks would allow for the introduction of diverse functional groups without the concerns of interfering with the biological folding patterns icquircd for biologically based systems While there have been reports of peptidomimetic compounds with defined secondary structure, these molecules do not generally self-assemble in biological solutions such as water.

Proteins and nucleic acids arc known for their extensive range of functions. This wide variety of functions depends on the biological macromolecule having the correct secondaiy structuie. This secondary stiucture is determined, in large part, by the primary sequence of the molecule, for instance, the amino acid sequence of a protein While the introduction o( extraneous functional groups is possible with biologically-based systems, if these groups disi upt the folding pattern of the molecule, the molecule will not function in the desired manner. Furthcrmoic, in oidei for these compounds to 1 unction in a therapeutic capacity the folding pattern must be maintained in biological solutions such as watei

Therefore, the synthesis and characterization of the large molecular structures of the present invention, that allow for the placement ot f unctional groups independent of then loldmg properties would allow foi fundamental questions legarding the ef fect ot these groups in biological systems to be addiessed Peptidomimetic compounds have been shown to be effective therapeutic agents, foi example as enzyme inhibitoi s (Rawls, 1996). Therefore, def ining the association of constiucts with def ined secondaiy structuies into larger assemblies, and the effect of functional groups on targeting these biomimetic molecules to precise locations within a cell, such as a lipid bilayei . could contribute to the to development therapeutic agents

AEDAMers aie def ined as large synthetic molecules exhibiting well-defined higher order stiucture based on a novel and entirely intiamolecular abiotic secondaiy structure

The abiotic secondary structuie derives f rom Alternating Electronic Donoi -Acceptoi interactions (Hunter & Sanders, 1990, Ashton et al.. 1991 ); hence, these new molecules aie called AEDAMeis. The AEDAMer structure involves an alternating array of electron rich

and electron deficient units, attached to each othei via a specially designed linking chain (FIG 1 ) The iesulting structuie has a cential coie ol stacked residues, sunounded by the functionahzed linking chain that is held ngidly in place While donoi -acceptor interactions have been used to effect the self-assembly of distinct molecules using intermoleculai interactions (Amabihno et al , 1995, Hunter & Sandeis, 1990), the present invention dcmonstiates the effectiveness ol donoi -acceptoi inteiactions in an intramolecular mannei to determine the secondary structure of complex synthetic molecules in solution This construction allows the folding propeities of the AEDAMei compounds to be distinct from the functional groups placed on the linkei

By changing the nature of the of othei groups on the linking unit the propeities and functions ot the entne complex can be controlled without affecting the ovciall lolded structuie

The AEDAMeis ot the piesent invention aie paiticulai ly promising foi potential drug applications These compounds may be used tot example, as anticancei agents. HIV inhibitors and immunoregulators Compounds may be developed to bind tightly the active site of vanous enzymes to cicate potent inhibitois In a similai mannei , compounds may be developed as icceptor ligands The compounds ol the piesent invention also may be used as lmmunotheiapeutic agents Compounds may be designed to mimic the stiuctuie ol a desired antigen which may not be appropnate lot dnect immunization For example, biological molecules which may not produce an immune icsponse in an individual due to toleiance may be imitated Also, pathogenic compounds such as viral proteins may be modeled and introduced into an individual to provide a foim of passive immunization Antigenic groups may be introduced into the linkei to enhance antibody production oi alternatively the linkei can be designed to be invisible to the immune system if desned The AEDAMei compounds may also be designed as earner proteins foi hapten piesentation to an individual

Due to the synthetic techniques foi producing AEDAMei s, a combinatonal approach may be taken whcie the ciystal structure of the target en/yme is unavailable

Also contemplated by the present invention are compounds where the AEDAMer structure is contained within an existing peptide or protein sequence. This allows for the properties of existing proteins to be altered or enhanced by the appending of an AEDAMer, for example, improving the transport of a peptide or protein across a membrane or improving specificity for the substrate or inhibitor of the protein. The β- sheet compounds provided by the present invention are particularly suited for this approach as many enzymes, such as proteolytic enzymes like serine proteases, bind their substrate as a β-pleated sheet and the signal peptides of most eukaryotic organisms are also active as a β-pleated sheet.

It long has been appreciated that electron transfer plays an important role in biological systems. A large molecular scaffold with a well-defined structure would provide an opportunity for the study of redox active functional groups in a well-defined orientation. It recently has been suggested that electron transfer has a significant role in the triggering of protein folding (Pascher et ai . 1996). The system of the present invention would allow the redox groups to be precisely arranged to mimic different arrangements during the folding process of a particular protein, such as cytochrome c. This would serve to elucidate the redox potential associated with virtually any state of unfolding in a manner not even possible with the fastest time scale experiments currently available.

There continues to exist a need for improved electro-optic polymeric systems. Electro-optic polymers have been known in the art for more than ten years. Since then, considerable effort has been devoted to producing electro-optic poly mers with long term stability. The engineering of these molecules into practical devices depends on producing stable molecules with the desired electro-optical properties. Electro-optic polymers contain a nonlinear optical (NLO) chromophore. The chromophore contains an electron donating group and an electron accepting group. These chromophores arc currently incoφorated into a matrices in a variety of ways including host guest systems where the guest chromophore is simply dissolved in the guest polymer. However, it has been shown that better results are achieved when the chromophores are covalently

attached to the polymer backbone This currently is achieved using classic polymerization techniques with extra stability introduced in the foim of crosslinking The piesent system, which would allow for the defined orientation of chromophores and precise placement of side chains within the polymei using lacilc synthetic techniques, would offer a significant advance to the field

Furthei modifications contemplated include AEDAMer compounds stable in organic solvents, amphipathic AEDAMeis that will fold into even laigei assemblies. AEDAMeis designed to insert into a membianc staictuie and the use of an AEDAMei compound as a scaffold foi electron transfer between metal complexes as piovided in

Example 8 While the basic AEDAMei structuie has the election donoi and electron acceptor alternating within the sequence, compounds also aie piovided with non-alternating sequences to allow the foimation of highei ordei sti uctuies such as knots and haiφins also desciibed in Example 8

C. DNA Intercalating Compounds (Polvatceptors)

Numerous molecules, including some clinically used chemotherapeuttc agents, intercalate into DNA a mode of mtei action that is chaiactei ized by inseilion of an planai group between base pans of the double helix DNA has as us two mam functions replication and tianscπption Any inteifeience with these piocesses has consequences to cellulai functions Theiefoie, DNA is well known as a taigct f oi di ug design Whereas most DNA inteicalating molecules contain a single inteicalating moiety (Lerman 1961 , Wilson & Jones, 1982), moie recently molecules with moie than one inteicalating group have been investigated (Wakelm, 1986, Denny et al 198^ Wu ih et al 1988 Atwell et al , 1983. Hansen et al , 1983, Laugaa et al 1985) An incieased af finity foi DNA and the largei number ot base pairs contacted by polyinteicalatois have in some cases, led to improved anti-tumoi activity (Wakelin, 1986 Waπng) and/oi sequence specificity (Waring 6c Fox, 1983, Delepiene et al 1991 ) iclative to then monointeicalating counteipaits While the limit to the numbei of inteicalating groups that can be positioned along a moleculai backbone is not known elloits to extend the mteiaction beyond

b/.s-intercalalion have been largely unsuccessful (Wakelin, 1986; Laugaa et ai. 1985). There arc two reasons for this. First, the previous syntheses of polyintercalators have been limited to time-consuming solution phase methods. The second inherent limitation to the traditional polyintercalating design is the connection of the intercalating units in parallel as substituents along a contiguous linker, similar to clothes hanging on a line.

The polyintercalators of the present invention incorporate solid phase synthesis techniques and DNA binding properties of a new scries of polyintercalators, including a tctraintcrcalating molecule, in which the aromatic groups are connected in series by polypeptide segments like beads on a string. A representative structure is provided in FIG. 4A and FIG. 4B showing two possible modes of intercalation. These polyintercalators are designed to be the first class of DNA-binding molecules in which desirable properties such as sequence specificity can be developed in a general way using combinatorial methods and/or rational design to change the sequence of the linker segments.

The compounds of the present invention provide the opportunity for constructs where the polyintercalator has properties of recognition based on DNA sequence specificity and a functional group such as an alkylating agent to modify the target DNA. Due to the fact that the polyintercalators of the present invention can be constructed beyond bw-intercalators. these compounds can be constructed to deliver more than two damaging agents to the target DNA. For example, bis or higher alkylation of a DNA sequence within a tumor would be expected to have an improved therapeutic value.

These polyintercalating molecules arc based on a modification of the AEDAMer compounds described above. In a particular embodiment, the polyintercalators of the present invention, use electron-deficient moieties to intercalate into double-stranded DNA (Yen et al.. 1982). The methods for synthesis of modified electron acceptors and an amino acid linker are provided in Example 3 and shown in Scheme 3. The synthesis of a representative polyintercalating molecule is provided in Example 6.

D. Polydonor Molecules

Some proteins have in their stiuctuie two oi moi e distinct polypeptide chains also known as subunits These polypeptide chains may be identical or may have diffeient sequences and structures entirely A well known example of a multimeric protein is hemoglobin, howevei , the association of subunits within a multimeric structuie can provide a vaπety of functions Foi example, the association of enzyme complexes aie used in metabolic pathways nbosomes are a complicated complex for protein synthesis and histones aie used in a stiuclui al role of nucleosomes In geneiai the subunits ot these multimeric stiuctutes aie associated by multiple non covalent interactions

Constructs wheie the monomei units all aie electi on-nch moieties also aie contemplated by the piesent invention These election donating molecules could combine with the electron deficient constructs desciibed abov e, to cieate a vast aπay ol highei oidei stiuctuie using the inteπnoleculai donoi acceptoi icactions between the compounds The two compounds may have difteient functional gioups on the linkei so that when the higher oidei molecule is formed the f unctional gioups aie displayed in a specific array accoiding to the desired function In this way, compounds may be produced as biomimetics ol multimeπc proteins

The methods aie provided foi the synthesis of the donoi monomeis in Example 2 and shown in Scheme 2 These donor monomei s may then be used to produce a poly¬ donor compound according to the methods provided in Examples 4 and 6

E. Synthetic Techniques

The compounds of the piesent invention may be synthesized using known methods foi peptide synthesis (Atheiton & Shepaid, 1989) The prefened method foi synthesis is standard solid phase methodology, such as that based on the 9- fluoienylmethyloxycarbonyl FMOC protecting group (Bai los et al , 1989) with glycine - tunctionahzed o-chloiotnty! polystyi ene icsm Methods loi synthesis of these

compounds are provided in Examples 1 , 2 and 4. These methods are also adaptable to placement of linking units on the end of the compound to provide additional functionalities, as desired.

A particular advantage to the solid phase method of synthesis is the opportunity for modification of these compounds using combinatorial synthesis techniques. Combinatorial synthesis techniques are defined as those techniques producing large collections or libraries of compounds, such as AEDAMeis, polyacceptors or poly-donors by sequentially linking different building blocks. Libraries can be constructed using compounds free in solution, but preferably the compound is linked to a solid support such as a bead, solid particle or even displayed on the surface of a microorganism. Several methods exist for combinatorial synthesis (Holmes et al., 1995; Burbaum et ai , 1995; Martin et ai, 1995; Freier et ai , 1995; Pei et ai , 1991 ; Bruce et ai . 1995; Ohlmeyer et ai, 1993); however, the preferred methods are split synthesis or parallel synthesis.

Split synthesis may be used to produce small amounts of a relatively large number of compounds, while parallel synthesis will produce larger amounts of a relatively small number of compounds. In general terms, using split synthesis, compounds are synthesized on the surface of a microparticle. Al each step, the particles are partitioned into several groups for the addition oϊ the next component. The different groups are then recombined and partitioned to form new groups. The process is repeated until the compound is completed. Each particle holds several copies of the same compound allowing for facile separation and purification. Split synthesis can only be conducted using a solid support.

An alternative technique known as parallel synthesis may be conducted either in solid phase or solution. Using parallel synthesis, different compounds are synthesized in separate receptacles, often using automation. Parallel synthesis may be conducted in microtiter plate where different reagents can be added to each well in a predefined manner to produce a combinatorial library. It is well understood that many modifications

of this technique exist and can be adapted foi use with the piesent invention A pieferied method foi synthesis of an AEDAMei combinatoi ial libi ai y is conducted using split synthesis and introducing an an ay of linking units between piedeteπnined monomei units to exploic the effect of diffeient functional gioups introduced onto the linkei Using combinatorial methods, variations in the acceptor units, donoi units and linking units may be introduced in a variety of combinations as pi ovided in Examples 9 and 10 Similaily, in the polyacceptor and polydonoi compounds the monomeis and linking units may be varied to produce a wide variety of compounds

F. Characterization

AEDAMei compounds ot the piesent invention also ottci an adv antage in characterization ot the folded structuies The aiomatic electronic donoi -acceptor interactions of the coie give use to hypochiomic absoi ption changes and visible legion charge transfei bands This provides a spectroscopic tool loi the initial chai actei ization and detection of piopei ly iolded stiuctuies This is pai ticulai iv useful when evaluating products from a combinatorial libraiy. as a piopci lv f olded sti uctuie will produce a chaiactenstic chaigc transtci band foi detection Details ot the chai actei ization ol an AEDAMei compound aie presented in Example 7

Foi J polv acceptor molecule designed loi DNA intcicalation, a number of different studies can be earned out to investigate the DNA-binding behavioi of the piedicted polyinteicalatois For example, inteicalating spec ies aie known to exhibit hypochiomism when bound to DNA due to the stackme intei actions piesent in the bound complex (Wakehn 1986), this also provides a lelatively simple sci een foi inteicalating compounds produced from a combinatoiial libraiy Details f or the chai acteπ/ation of DNA binding compounds aic piesented in Example 4

G. Therapeutic Methods

In accordance with the present invention, numerous therapeutic methods are contemplated in which compounds of the present invention will be employed. Practically any therapeutic endeavor that relies on a therapeutic biological molecule can be adapted for use with a synthetic compound. The advantages that the instant compounds bring to any theiapy may be considerable - target selectivity lack of toxicity. inci eased stability and/or half-life in vivo and increased activity.

In one embodiment, the compounds ot the present invention may be used in traditional chemotheiapeutic protocol for the treatment of cancels In theory, piactically any chemotherapeutic can be mimicked by the appropriate synthetic molecule Chemotherapeutics that would be used as functional models are alkylating agents (e.g., nitrosourcas, ethylemmines, alkylsullonates), cytokines ά_ growth tactoi s {e g , IL-2. GM-CSF. gamma-IF), steroid hormones (e.g , estrogen, piogestins, andi ogcn-,), antimetabohtes (e.g , purine analogs, pyrimidine analogs, f olinic acid analogs) oi antibiotics (e.g., mitomycin).

In particular the present invention can provide compounds that inteicalale with DNA. This ability may be exploited in two ways Fu st, the meie piesence in DNA of certain intercalated compounds can interfere with the activities ot both DNA and RNA polymerases. This may introduce lethal changes in the DNA and RNA produced by these enzymes or it may simply prevent their function In both cases, it is anticipated that cell death will occur It the amount of cell death is equivalent to the growth i ate ot the tumoi , tumor progiession may be halted. If the amount of cell death is greater than the growth rate of the tumor, tumor eradication may be achieved

Second, it may prove useful to employ bifunctional compounds of the piesent invention that not only target DNA, but actively damage the DNA once targeted Such bifunctional compounds may be administered in an active form or as a prodrug that is activated in the body by natural or provided agents One example ol such a method

would involve the attachment of a DNA-damaging agent to an intercalating moiety. Once the DNA has been targeted by the intercalator, the DNA-damaging agent can exert its action on the DNA.

In another embodiment, the compounds of the present invention may be used as immunogens. The modeling of antigenic epitopes on various molecules, e.g., proteins, lipids, carbohydrates or steroids, may be undertaken using compounds of the present invention. The technology may be applied merely in the production of antisera to these molecules in experimental animals. The cost, quantity and purity of synthetic compounds may prove superior to the natural compounds. They also may be employed as adjuvants that stimulate the immune response, or as inert but highly stable carriers that increase the half-life of other compounds, i.e.. haptens. without themselves being immunogenic.

On the other hand, immunogenic compounds of the present invention may be used as vaccines to raise antibodies against pathogenic agents such as bacteria, viruses or tumor antigens. Alternatively, vaccines based on the present invention can be used to tolerize patients to therapeutic compounds that normally would elicit a significant (and perhaps deleterious) immune response. Another type of vaccine would involve the use of electron-donor, electron-acceptor molecules to raise an immune response against some naturally occurring compounds, e.g.. a hormone or receptor, such that the immune response will down-regulate the action of that natural compound.

Yet another therapeutic method involves the use of the instant compounds as targeting agents. One of the major limitations ol ^' in vivo therapies is the inability to target therapeutics to appropriate locations within the patient. For example, while gene therapies permit exquisitely specific molecular intervention, the ability to target this intervention to only those cells that require it has proved problematic. By generating compounds that are able to target specific cells, for example, by specific binding to cell surface receptors or tissue marker, it is possible to deliver therapeutics to cells in a much

more specific fashion The therapeutic may be a traditional cancel chemotheiapeutic, an antibiotic, a toxin, a gene vectoi , a hormone oi othei agent

In a similar manner, compounds accoiding to the piesent invention can be used similai to receptor ligands Foi example, it may be desirable to stimulate receptors in vivo (l) where insufficient ligand exists in the subject oi (n) wheie the leceptors aie part of a closely related family but only one of the family is to be activated By engineering a compound that will bind the ligand of inteiest, one can selectively cause a hgand-specif ic stimulation of the ieceptoi A synthetic species that acted in the same manner as human growth hoimone oi human insulin would have tremendous benef it in this regard

In still yet another embodiment one may use the compounds of the piesent invention in a therapeutic method wheie the compound is itself active This might be achieved by ci eating an molecule that mimics the active site of an en/yme Enzymes that might be used as models foi iational design are HGPR I (gout) galactose- 1 -phosphate uπdyltiansferase (galactosemia), glucose-6-phosphate t,von Gieike's disease), lysosomal glucosidasc (Pompe s disease), muscle phosphoiylase (McAidle' s syndrome) glucoceiebosidase (Gauchei 's disease), α-L-iduionidase (Hui lei syndrome), L-iduionate sulfatase (Huntei syndrome), sphingomyehnase (Niemann Pick disease) and hexosamimdase (Tay-Sachs disease)

Finally, it may be desirable simply to modify existing thei apeutic compounds according to the present invention to improve then f unction Such an improvement might be achieved by incieasing the existing compound's stability oi longevity in vivo Alternatively it may piove useful to use compounds of the present inv ention to mask the antigenic characteristics ot the existing compound from the host's immune system It is likely that one will look both the kind of synthetic addition and its point of attachment to the existing molecule when developing such an approach

H. Pharmaceutical Compositions

Pharmaceutical compositions of the piesent invention comprise an effective amount of the expiession construct dissolved 01 dispeiscd in a phaimaceutically acceptable carrier, such as a pharmaceutically acceptable buffer, solvent or diluent, or aqueous medium Such compositions also can be leleπed to as inocula.

The phrases "pharmaceutically oi phaimacologically acceptable tefers to molecular entities and compositions that do not pioduce an adverse, allergic oi other untoward icaction when administered to a human As used herein the terms

"phai maceutically acceptable carπei " and "pharmaceutically acceptable buffei . solvent oi diluent" include any and all solvents, dispersion media, coatings, antibactenal and antifungal agents, isotonic and absoiption delaying agents and the like The use of such media and agents foi pharmaceutical active substances is well known in the ait Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the theiapeutic compositions is contemplated Supplementary active ingredients can also be incoφoiated into the compositions

In addition to the compounds formulated loi pai entei al administration, such as intiavenous oi intramusculai injection, othei phai maceutical ly acceptable loi ms include, e.g., tablets or othei solids for oial administiation, time lelease capsules, and any othei form currently used, including cremes, lotions, moulhwashes. inhalents and the like

I. Routes of Administration

As used heiein the terms "contact", "contacted ', and "contacting", are used to describe the process by which an effective amount of a phaimacological agent, e.g.. the expiession constructs disclosed in the piesent invention, comes in dnect juxtaposition with the target cell.

For methods of treating mammals, pharmaceutical compositions may be administered by a variety of techniques, such as parenteral, topical or oral administration.

For example, the active constructs also be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub¬ cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains an expression construct agent as an active ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon the addition of a liquid prior to injection can also be employed; and the preparations can also be emulsified.

Solutions of the active constructs as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in giycerol. liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevenl the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The expression construct can also be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic.

tartanc, mandelic, and the like Salts formed with the fiee carboxyl groups can also be deπved from inorganic bases such as, loi example, sodium, potassium, ammonium, calcium, oi ferric hydroxides, and such organic bases as isopiopylaminc. tiimethylamine, histidine, procaine and the like

The earner can also be a solvent or dispeision medium containing, foi example, water, ethanol, polyol (loi example, giycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils The propei fluidity can be maintained, foi example, by the use oi a coating such as lecithin, by the maintenance of the required particle size in the case of dispeision and by the use ol sui factants The prevention of the action of nπcrooiganisms can be brought about by vaπous antibactenal ad antifungal agents, foi example, parabens, chloiobutanol phenol sorbic acid, thimeiosal, and the like In many cases it will be pieterable to include isotonic agents, foi example, sugai s oi sodium chloπde Prolonged absorption of the ιn)ectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin

Sterile injectable solutions aie prepaied by incoipoiating the active constructs in the rcquned amount in the appropriate solvent with vanous of the othei ingredients enumeiated above, as lequned, lollowed by filteied steπhzation Generally, dispeisions are prepaied by incoipoiating the vanous steπhzed active ingiedients into a sterile vehicle which contains the basic dispersion mediαm and the rcquned othei ingredients from those enumeiated above In the case of sterile powdcis foi the piepaiation of sterile injectable solutions, the prefened methods of pieparation aie vacuum-drying and fieeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a pieviously steπle-filteied solution thereof

Upon formulation, solutions will be admimsteied in a mannei compatible with the dosage toimulation and in such amount as is therapeutically effective The formulations aie easily administered in a variety of dosage forms such as the type of

mjectable solutions described above, but drug ielease capsules and the like can also be employed

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessaiy and the liquid diluent first rendered isotomc with sufficient saline or glucose In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure Foi example, one dosage could be dissolved in 1 mL ol isotomc NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (sec for example, "Remington s Pharmaceutical Sciences' 15th Edition, pages 1035- 1038 and 1570-1580) Some variations in dosage will neccssaπly occui depending on the condition of the subject being treated The pcison responsible for administration will, in any event, determine the appropπate dose lor the individual subject

The lollowing examples aic included to demonstiate prefened embodiments of the invention It should be appicciated by those of skill in the ait that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to f unction well within the practice of the invention, and thus can be considered to constitute piefeiied modes foi ol practice Howevei, those ot skill in the art should, in light of the piesent disclosuie. appieciatc that many changes can be made in the specific embodiments which aic disclosed and still obtain a like oi similar result without depaiting from the spirit and scope of the invention

EXAMPLE 1

Synthesis of Preferred Acceptor Residues of AEDAMer

FMOCN-Asp(t-Bu)-Acceptor-OH (12) Compound 11 (1 0 g, 2 1 mmol) was suspended in 10 mL CH ₂ C1 ₂ and 10 mL TFA was added slowly After standing for 10 min , the solution was evapoiated and the residual TFA removed by evaporation from

MeOH The iesulting white solid was tntuiated with cthei, hlteied, and dπed in vacua to

yield 1.04 g (2.1 mmol, 100%) of the deprotected amino acid as its trifluoroacetate salt. This solid was suspended in DMF (50 mL) and FMOC-Asp(OBut)-OPfp (Advanced Chemtech, 1.15 g, 2.00 mmol, 0.95 eq. ) was added, followed by 1-hydroxybenztriazolc (HOBT, 0.283 g. 2.10 mmol) and 2,6-lutidinc (0.899 g. 8.40 mmol. 4 eq. ). The reaction mixture was stirred overnight and then partitioned between ethyl acetate and sodium citrate buffer (0.2 M, pH 4.5). The aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the combined organic layers were washed with citric acid buffer once, then with brine (3 x 50 mL), dried over anhydrous Na ₂ Sθ _L filtered, and evaporated in vacuo. The resulting solid was triturated with Et ₂ 0 (2 x 30 mL). filtered, and dried under vacuum to yield 1.50 g 12 ( 1 .93 mmol. 93%). Η NMR (300 MHz, DMF-c/ ₇ ) d 8.68 (s,

4H), 8.22 (t, l H, 7 = 5.8 Hz), 7.88 (d, 2H, 7 = 7.4 Hz). 7.65 (dd, 2H. ./ =7.2. 2.9 Hz), 7.56 (d, 2H, .7 = 8.3 Hz), 7.41 (dt, 2H, 7 = 7.3. 3.8 Hz), 7.28 (dt. 21 1, 7 = 7.3 Hz). 4.46-4.28 (c, 5H), 4.20-4.08 (c, 2H), 3.63 (dt, 2H, J = 5.7 Hz), 2.75 (t. 211. ./ = 7.8 Hz), 2.58 (dd, I H, 7 = 9.2, 16 Hz), 1.37 (s, 9H); ^H C NMR (300 MHz, DMlw/ ₇ ) d 1 71 .6, 170.4. 169.0. 162.2, 161.5, 155.3, 143.4. 140.2, 129.6, 129.5, 126.9, 126.3. 125.6, 124.5, 1 19.2. 79.3, 65.6.

51.3, 46.1 , 39.1 , 36.9, 36.4, 35.6, 31. 1 , 26.6, HRMS (FAB ) 775.2608 (775.2615 calcd for C ₄₂ H ₁₉ N ₄ O| , . MH).

FMOCN-Asp(t-Bu)-Acceptor-OPfp (13) Compound 12 ( 1.50 g, 1 .93 mmol) was dissolved in DMF ( 15 mL) and to it was added pentafluorophenol (0.53 g, 2.9 mmol, 1.5 eq.) followed by dicyclohexylcarbodiimide (0.44 g. 2. 1 mmol. 1 . 1 eq.). The reaction was stirred overnight and then evaporated under high vacuum to a brown sludge. 1 ,4-

Dioxane (50 mL) was added and the mixture stirred for 2 h. The precipitate was removed by filtration and evaporated under high vacuum to a yellow solid. This was triturated with pentane (2 x 50 mL), filtered, and evaporated in vacuo to yield 13 ( 1 .78 g.

1.89 mmol, 98%) as a yellow-orange solid. Η NMR (300 MHz, CDC1 _. 0 d 8.61 (s, 4H),

7.73 (d, 2H, 7 = 7.3 Hz). 7.58 (d, IH, J = 7.3 Hz), 7.53 (d. I H, ./ = 7.3 Hz). 7.4-7.3 (m,

4H), 6.94 (br t, I H). 5.75 (d, I H, ./ = 13 Hz), 4.59 (t. 2H. ./ = 7.2 Hz), 4.5-4.25 (m. 511),

4.05 (t, IH, 7 = 7.0 Hz). 3.9-3.6 (m, 2H), 3.14 (t, 2H. 7 = 7.3 Hz), 2.81 (dd. I H. 7 = 4.4, 17 Hz), 2.45 (dd, 1 H. 7 = 5.4, 17 Hz), 1. 14 (s, 9H); ' 'c NMR (300 MHz. CDCLW 171.0,

166.8, 163.2. 162.4. 156.0, 143.5, 141.1 . 131.0. 127.7. 127.0. 126.0, 124.9, 1 19.9, 81 .4,

67.1, 39.8, 38.9, 36.8, 35.9, 31.5, 27.6; HRMS (FAB) 941.2472 (941.2457 calcd for C ₄₈ H^F,N,0,ι).

Synthesis Of Acceptor Residues

H ₃ N ⁺ (CH2)2C0 ₂ Bn(TsO-) H ₂ N(CH ₂ ) ₂ NBOC 1.H ₂ , Pd/C,

Et(/-Pr) ₂ N, i-PrOH, reflux 2 Chromatogr oaphic Separation

Statistical Mixture: A: Ri = R ₂ = (CH ₂ ) ₂ C0 ₂ Bn B: R, = R ₂ = (CH ₂ ) ₂ NBOC C. R , = (CH ₂ )2C0 ₂ Bn, R ₂ = (CH ₂ )2NBOC

Ri =(CH ₂ ) ₂ C0 ₂ H R ₂ = (CH ₂ ) ₂ NBOC 40%, two steps

11 3.

Scheme 1

EXAMPLE 2 Synthesis of Preferred Donor Residue for AEDAMer

l-(N-tert-butoxycarbonyl-3-aminopropylxy)-5-benzyloxynaph thalene (5). l-benzyloxy-5-hydroxynaphthalene (4.02 g, 16.1 mmol) was combined with

BOCN(CH ₂ ) ₃ Br (3.83 g, 16.1 mmol) in 125 ml DMF and the solution was purged with argon. CS2CO3 (6.55 g, 20.1 mmol) was added and the solution was heated to

75°C for 15 h. The DMF was removed by vacuum distillation and the resulting residue was partitioned between saturated aqueous NaHCθ3 and CH2CI2 (3 x 50 ml). The combined organic layers were washed with saturated aqueous NaHCθ3 (3 x 50 ml) and with brine (3 x 50 ml), dried over anhydrous Na2Sθ4, filtered, and evaporated in vacuo. The resulting solid was recrystallized with hot methanol to yield 5 as light brown needles (4.59g, 1 1.3 mmol, 70%). Η NMR (300 MHz, CDCU) δ 7.93 (d, I H, 7 = 8.5 Hz), 7.84 (d, IH, 7 = 8.5 Hz), 7.54-7.24 (c, 6H), 6.91 (d. 7 = 7.5 Hz), 6.83 (d. I l l, 7 = 7.6 Hz), 5.23 (s, 2H), 4.95-4.85 (br, I H), 4.18 (t, 2H, 7 = 5.8 Hz), 3.42 (q, 2H, 7 = 6.2 Hz), 2. 10 (p, 211, 7 = 6.2 Hz), 1.45 (s. 9H); ⁿ C NMR (300 MHz, CDC1 ₃ ) 5 156.0. 154.3, 154.2, 137.1 , 128.5, 127.8, 127.3, 126.8, 126.7. 125.1 , 1 14.6, 1 14.4, 106.0, 105.4, 79. 1 , 70. 1 , 66.2, 38.3, 29.5, 28.4; HRMS (FAB) m/z 407.2105 (407.2096 calcd for C25H29NO4. MH).

l-(N-ttτt-butoxycarbonyl-3-aminopropyloxy)-5-hydroxynaph thalene (6). A solution of 5 (4.59 g, 1 1.26 mmol) in 250 ml of CHCI3/MCOH ( 1 : 1 ) was purged with argon and to it was added 1 g of 10% Pd/C. The solution was purged with H2 and stirred for 10 h, after which it was filtered through a short pad of celite and concentrated in vacuo to yield 3.14 g (9.89 mmol, 87%) of 6 as a light brown solid. 'II NMR (300 MHz, CDCh) 57.80 (d, IH, 7 = 8.5 Hz), 7.76 (d, I H, 7 = 8.6 Hz), 7.36 (t, I H, 7 = 8.1 Hz), 7.27 (1, I H, 7 = 7.9 Hz), 6.86 (d, I H. 7 = 7.5 Hz), 6.83 (d. I H, 7 = 7.6 Hz), 5.0-4.8 (br. 111), 3.44 (t, 2H, 7 = 5.8) 2.12 (p, 2H, 7 = 6.1 Hz), 1.45 (s, 9H); ⁿ C NMR (300 MHz, CDC1-,) <5 156.4, 154.2, 152.0, 126.9, 125.7. 125.3, 124.8, 1 14.3,

1 13.8, 109.3, 105.1 , 79.6, 66.2, 38.5, 29.4, 28.4; HRMS (FAB) m/z 317.1628 (317.1627 calcd for C, ₈ H ₂₃ N0 ₄ . MH).

l-(N-tert-butoxycarbonyl-3-aminopropyloxy)-5-(3-benzyloxy carbonyI propyloxy)-naphthalene (7). Compound 6 ( 1.0 g, 3.15 mmol) was combined with benzyl 4-bromobutyrate ( 1.22 g, 4.73 mmol) in 25 ml DMF and the solution was purged with argon. CS2CO3 ( 1.28 g, 3.94 mmol) was added and the solution was stirred at 60°C for 17 h under argon. The DMF was distilled from the reaction under high vacuum such that the temperature was not allowed to exceed 60°C. The resulting residue was partitioned between EtOAc (25 ml) and brine (25 ml) and the layers were separated. The aqueous phase was extracted with EtOAc (3 x 25 ml ) and the combined organic layers were washed with brine (3 x 25 ml) and saturated aqueous NaHCθ3, dried over anhydrous Na2Sθ4, filteted. and concentrated in vacua.

Purification by silica gel flash chromatography (CHoCh → 5% MeOH/CHoC'b) gave 1.20 g (77%) 7 as an off-white solid. Η NMR (300 MH/, CDCN) 5 7.84 (d, 1 HJ = 8.4 Hz), 7.82 (d. IH, 7 = 8.28 Hz), 7.4-7.3 (c, 711). 6.83 (d. 1 H, 7 = 6.2 Hz). 6.81 (d. IH, 7 = 7.1 Hz), 5.14 (s, 2H), 4.9 (br s, I H), 4.20 (t. 211. 7 = 5.4) 4.17 (t. 2H, 7 = 5.7), 3.43 (br dt, 2H). 2.69 (t, 2H, 7 = 7.2 Hz), 2.28 (p, 211. 7 = 6.4Hz). 2.12 (p, 2H. 7 = 6.0 Hz), 1.45 (s, 9H) : ^π C NMR (300 MHz, CDCh) <5 172.8. 155.9. 154. 1. 135.7. 128.3. 127.9, 127.6, 127.1 , 126.4, 1 14.3, 1 14. 1 , 109.0, 78.9. 69.8. 66.6, 38.1 , 30.8, 29.3. 28.2, 24.5; HRMS (FAB) m/z 493.2453 (493.2464 calcd for C ₂ qH„N0 ₆ , MH).

l-(N-tert-butoxycarbonyl-3-aminopropylo.\y)-5-(3 ^, -carboxypropyloxy) naphthalene (8). Compound 7 (3.13 g, 6.35 mmol) was dissolved in 125 ml EtOH and the solution was purged with argon. 1.01 g 10% Pd/C was added to the reaction vessel and the atmosphere was replaced with H ₂ . After 3 h. TLC (5% MeOH/CH ₂ Cl ₂ ) showed that the reaction had gone to completion. The reaction was diluted with CH ₂ C1 ₂ and filtered through celite. The filter was rinsed with CH ₂ CI ₂ /MeOH/AcOH (80: 15:5) until the rinsings were no longer UV-active. The filtrate was concentrated //; vacuo to a volume of 15 mL, and /-Pr ₂ 0 was added to crystallize the product, 8. as

white needles (2.45 g. 96%). Η NMR (300 MHz, 10% CD ₃ OD/CD ₂ CI ₂ ) 5 7.78 (d, 2H,7 = 8.4 Hz), 7.30 (t, 2H, J = 8.0 Hz), 6.81 (d, 2H, 7 = 7.6 Hz), 4.12 (t. 14H, 7 = 5.9 Hz), 3.31 (t, 2H, 7 = 6.7 Hz), 2.56 (t, 2H, 7 = 7.3 Hz), 2.17 (p, 2H, 7 = 6.6 Hz), 2.04 (p, 2H, 7 = 6.3 Hz), 1.38 (s, 9H) ; ¹³ C NMR (300 MHz, 10% CD ₃ OD/CD ₂ Cl ₂ ) 5 176.7, 156.9, 155.1, 127.3, 125.7, 1 14.8, 1 14.7, 106.0, 79.7, 67.8, 66.7, 38.6, 31.3, 30.1 , 28.5, 25.3; HRMS (FAB) m/z 403.1997 (403.1995 calcd for C ₂₂ H _2g N0 ₆ , MH).

Preparation of "FMOC-Asp-Donor-OH" (9). Compound 8 (0.50g, 1.24 mmol) was dissolved in 10 ml TFA/CH ₂ C1 ₂ ( 1 : 1 ) and stirred for 15 min. The reaction was evaporated to a green solid and residual TFA was removed by evaporating several times from MeOII (3 x 25 ml). The resulting amino acid was converted into its triethylammonium salt by dissolving the residue in MeOI I ( 10 ml) and adding 2 ml TEA. The resulting suspension was sonicated for 30 seconds and evaporated. The solid was suspended in Et ₂ 0, filtered, rinsed with ether, then pentane, and dried ; ^' /; vacuo to yield 0.59 g (0.96 mmol, 11%) of a white solid that was immediately carried on to the next step without further purification or characterization. The solid amine was suspended in 50 ml dry DMF and to it was added FMOC-Asp(Of-Bu)-OPfp (Advanced Chemtech, 0.53 g, 0.91 mmol = 0.95 eq.) and HOBT (0.12 g, 0.91 mmol). After stirring for 2.5 h, the reaction appeared to be complete by TLC (5% MeOH/CH ₂ Cl ₂ . monitoring the disappearance of the baseline spot indicative of the amino acid starting material). The reaction was worked up by partitioning between 0.2 M Na citrate buffer (pH 4.5, 100 ml) and EtOAc ( 100 ml). The aqueous layer was extracted with EtOAc (3 x 50 ml) and the combined organic layers were washed with brine (3 x 50 ml), dried over anhydrous NaS0 ₄ , filtered, and evaporated in vacuo. The resulting solid was taken up into EtOAc and then precipitated by the slow addition of pentane. The precipitate was filtered, dried in vacuo, and the residual HOBT was removed by triturating the product several times with Et ₂ 0. After drying in vacuo overnight, 9 (0.474 g, 77%) was obtained as an off-white solid. Η NMR (300 Mhz, DMF-J ₇ ) 5 8.26 (br t, I II), 7.90 (d, 2H, 7 = 7.4 Hz), 7.82 (d, 2H, 7 = 8.3 Hz), 7.72 (t, 4H, 7 = 6.9 Hz), 7.47-7.30 (m, 511), 6.98 (t, 2H, 7 = 8.2 Hz), 4.59 (dt, I H, 7 = 7.6, 5.9 Hz). 4.35-4.2 (m, 5H), 3.52 (br dt. 2H). 2Λ) l (dd, I H, 7 = 5.5, 16 Hz), 2.63

(t. 2H, 7 = 7.4 Hz), 2.3-2.1 (m, 4H), 1.42 (s, 9H); ^π C NMR (300 Mhz, DMF-;/ ₇ ) 174.8. 171.5, 171.0. 170.5, 156.8, 155.1 , 155.0, 144.8, 144.7, 141 .7. 128.3, 127.7, 127.2, 127.1 , 126.0, 125.9, 120.7, 1 14.5. 1 14.4, 106.2. 80.8. 67.9. 67.0, 66.3. 52.8, 47.7, 38.6, 36.9, 31.1 , 28.0, 25.3, 20.8; HRMS (FAB) m/z.

Preparation of "FMOC-Asp-Donor-OPfp" (10). Compound 9 (0.75 g, 1.1 mmol) was dissolved in 10 mL dry DMF, and to it was added pentafluorophenol (0.297 g, 1.5 eq.), followed by dicyclohexylcarbodiimide (0.244 g, 1.1 eq.). The reaction was stirred overnight and the DMF was evaporated under reduced pressure. 1 ,4-Dioxane (25 mL) was added and the mixture was stirred for 3h. The precipitate was removed by filtration and the resulting solution was evaporated /// vacuo. The resulting yellow oil was triturated with pentane to pioduce a gelatinous solid, which was filtered and dried in vacuo to yield 10 (0.82 g, 0.96 mmol, 89% ). Η NMR (300 MHz, CDC1 ₃ ) 5 7.86 (d, 2H, 7 = 8.5 Hz), 7.8 1 (d. 2H, 7 = 8.5 Hz), 7.73 (d, 2H, 7 = 7.3 Hz), 7.5 l (d, 211, 7 = 7.3 Hz). 7.41 -7.25 (c, 611), 6.82 (dd, 2H, 7 = 7.6, 3. 1 Hz), 6.72 (br t, I H), 5.91 (d, IH, 7 = 8.1Hz), 4.48 (br, IH), 4.35 (d. 2H. 7 = 6.8 Hz), 4. 19 (t, 2H, 7 = 6.0 Hz). 4. 17 (t, 2H, 7 = 6.3 Hz), 4.12 (t, I H, 7 = 6.9 Hz), 3.55 (dt, 2H, 7 = 6.6, 17.0 Hz), 2.36 (p, 2H, 7 = 6.2 Hz), 2.13 (p, 2H, 7 = 6.2 Hz), 1 .43 (s. 9H); ^π C NMR (300 MHz. CDC1 ₃ ) 5 171.2, 170.6, 169.2, 154.3. 154. 1 . 143.7. 141 .3. 127.8. 127.1 , 126.7. 126.6, 125.2, 125.0 (two peaks). 120.0 (two peaks), 1 14.5, 1 14.3, 105.7. 105.5, 81.9, 67.1 , 66.3, 65.8, 51.3, 47.1 , 37.4, 37.3, 30.2, 29. 1. 28.0. 24.6; HRMS (FAB) m/z 862.2877 (862.2889 calcd for C ₄₆ H ₄₃ F ₅ N ₂ 0 ₉ , M).

Synthesis Of Donor Residues

EXAMPLE 3

Synthesis of Preferred Acceptor Residues for Polyintercalator

N-(2-tert-butoxycarbonylaminoethyl)-N'-(2-carboxyethyl)-l ,4,5,8- naphthalene tetracarboxylic diimide (17). 1 4,5 8-naphthalenetetracarboxyhc dianhydπde (8 96 g, 33 4 mmol) was suspended in 450 ml /-PrOH tei t- butoxycarbonylaminoethylamine (5 34 g, 33 4 mmol) and /3-alanιne benzvl ester p- toluenesultonate salt ( 1 1 72 g, 33 4 mmol) weie added to the mixture, followed by Ν Ν-dnsopropylethylamine (6 4 ml, 37 mmol) The mixtuie was heated to ieflux toi 24 h, after which it was concentrated in vacuo The resulting solid was partitioned between CH ₂ CL (450 ml ) and 0 1 M sodium citiate but ler (pH 4 5 500 ml ) The oi game layei was washed with citiate buffet (2 x 100 ml ) satuiated ΝaHCO , ( 3 x 100 ml), and then bπne ( 100 ml), and dned ovei anhydious ΝaSO ₍ MeOH ( 100 ml ) was added to the mixtuie and it was filtered thiough a shoi t pad of silica gel The Oltei was rinsed with 10% MeOH/CH ₂ CL ( 100 ml ) and the supernatant conccntiated to yield the expected statistical mixture of pioducts ( 18 7 g 91 % ) This mixtuie was earned on to the next step without fuithei put ideation

The abo\ e tan solid was suspended with sonication in absolute EtOH (250 ml) and purged with Ai 14 g \ 07o Pd/C was added f ollowed b\ 15 ml 1 ,4- cyclohexadiene added diopwise The mixtuie was chai ged with H ₂ at atmosphenc piessuie and stured toi 48 h Aftei concentiatmg the icaction to 50 ml (bath temp

30°C, 20 torr), 250 ml 1 5% TEA/CH ₂ C1 ₂ was added I he lesulting sluiry was stu red foi 20 mm and hlteied thiough celite The filtei was nnsed w ith 10 ^c /< TEA/CH ₂ C1 _^ until the rinsings weie coloilcss and the solution was evapoi ated m \ acuo I he resulting solid was taken up into CH-.CL with μist enough MeOH to allow lot complete solvation. and the product mixture was deposited onto silica by evapoiation

Chiomatographic separation using a giadient ol O→ l O^r MeOH in 10% TEA/CHoCl ₂ yielded 17 as the second, bπght orange band Altei conccnti ation the solid was taken up into 10% MeOH/CILCK and 20 ml HOAc was added Hexanes (300 ml ) was slowly added to piecipitate the pioduct Aftei cooling loi sevei al hours, the mixtuie

was filteied and the piecipitate was rinsed with MeOH and dried ;;; vacuo to yield 17 as (5 10 g, 27%) as a fine white powdei Η NMR (DMSO-c/ ₆ ) d 8 58 (s 4H) 6 90 (br t, III), 4 25 (t, 7=7 5 Hz, 2H) 4 12 (bi t, 2H), 3 27 (bi q, 2H), 2 62 (t / =7 4 Hz, 2H) 1 20 (s, 9H), ^π C NMR (DMSO-J ₆ ) 172 4, 162 7, 162 4, 155 8, 130 4, 130 3, 126 4, 126 1 , 77 5, 37 6, 36 1 , 32 0, 28 1 , HRMS (FAB) m/z 482 1547 (482 1563 calcd for C ₂₄ H ₂₄ N ₃ 0 ₈ )

N-2-(N'-9-fluorenylnιethoxycarbonylglycyl)-aminoethyI-N' -(2- carboxyethyl)-l,4,5,8-naphthalenetetracarboxylic diimide (18). Compound 17 (2 g, 4 16 mmol) was suspended in 15 mL CH ₂ C1 ₂ and 1 5 mL TFA was added slowly Aftei standing foi 10 min the solution was evapoi ated and the iesidual TFA removed by evapoiation tiom MeOH T he icsulting while solid was suspended in MeOH ( 100 mL) and T EA (50 mL) was added The mixtuie was sonicated foi seveial minutes, Et ₂ 0 ( 100 mL) was added and the lesulting solid was liltcied and dned m \ ac uo to yield 1 64 g (2 92 mmol, 70%) ol the depi otectcd amino acid as its fiethylammonium salt This solid was suspended in DMT (25 mL) and N 9- fluoicnylmethoxycai bonylglycinc pcntalluorophenyl estei ( 1 92 g, 4 16 mmol) was added followed by 1 -hydιoxybenztι ιazole (HOB 1 5 16 mg 4 16 mmol) and 2,6- lutidine (890mg, 8 32mmυl ) Alter stimng loi 4 h the mixtuie was vacuum filteied to lemove any piecipitate, the supernatant was pouted into H ₂ 0 (35 ml) and the resulting suspension was allowed to stand foi seveial houis I he mixture was filteied and the resulting yellow solid was rinsed with H->0 and dπed in the piesence ol P->0^ in a vacuum dessicatoi overnight The crude pioduct was tπturated with Et ₂ 0 several times (lo lemove the residual 1 -hydιoxybenztιιazole and pentafluoiophenol) filtered and dried in vacuo to yield 18 ( 1 79 g, 2 72 mmol, 65%) as a yellowish powdei Η ΝMR (300 MHz, DMF-J ₇ ) tl 8 66 (s, 4H), 8 12 (t, IH 7 = 5 7 Hz), 7 88 (d, 2H, / = 7 4 Hz), 7 68 (d, 2H, 7 =7 3 Hz), 7 4 (m, 1 H). 7 42 (t, 2H, 7 = 7 4 Hz) 7 30 (t, 2H, 7 = 7 3 Hz), 4 40 (t, 2H, 7 = 7 3 Hz), 4 3 1 (t, 2H, 7 = 5 6 Hz), 4 20 (d, 2H, 7 = 6 9 Hz), 4 1 1 (t, I H, 7 = 6 5 Hz), 3 75 (d, 2H, / = 5 8 Hz) 3 66 (bi dt, 211, J, = 5 5), 2 81 (t 2H, / = 7 7 Hz), ⁿ C ΝMR (300 MHz DMF-J ₇ ) tl 173 0, 170 4, 163 6, 162 9, 157 1

144.8, 141.6, 131.1, 131.0, 128.3, 127.7. 127.2, 127.0, 126.0, 120.6, 67.0, 47.5, 44.6, 40.6, 37.0, 36.2, 32.5; HRMS (FAB) 661.1934 (661.1934 calcd for C ₃₆ H29N4O9, MH).

N-2-(/V- 9-fluorenylmethoxycarbonyIglycyl)-aminoethyI-N'-(2- pentafluoro phenoxycarbonyl-ethyl)-l,4,5,8-naphthalenetetracarboxylic diimide

(19). Compound 18 (1.1 1 g, 1.68 mmol) was dissolved in dry DMF (5 mL) and pentafluorophenol (335 mg, 1.85 mmol), followed by dicyclohexylcarbodiimide (381 mg, 1.85 mmol), were added. After stirring for 16 h, the reaction was evaporated in vacuo at room temperature to a viscous sludge, to which was added dry dioxane (10 mL). The resulting suspension was stirred for 5 h and then filtered. The bright yellow filtrate was evaporated in vacuo and the resulting solid was triturated with pentane. The mixtuie was filtered and the product was rinsed with pentane and dried in vacuo to yield 19 ( 1.21 g, 1.47 mmol, 87%) as a yellow powder. Η ΝMR (300 MHz, DMF- th) d 8.67 (s, 4H), 8.13 (t, IH, 7 = 5.6 Hz), 7.86 (d, 2H, 7 = 7.4 Hz), 7.65 (d, 2H, 7 = 7.3 Hz), 7.47 (t, IH, 7 = 6.0 Hz). 7.39 (t, 2H, 7 = 7.3 Hz). 7.27 (t, 2H, 7 = 7.4 Hz), 4.55 (t, 2H, 7 = 7.1 Hz), 4.30 (t, 2H, 7 = 5.6 Hz), 4.16 (d. 2H, 7 = 6.8 Hz), 4.09 (d, IH, 7 = 6.4 Hz), 3.72 (d, 2H. 7 = 5.9 Hz), 3.63 (dt, 7 = 6.1 Hz). 3.35 (t, 2H, 7 = 7..2 Hz); ^{l 3} C ΝMR (300 MHz, DMF-J ₇ ) d 170.4, 168.4, 163.6. 162.9, 157.3, 144.8, 141.6, 131.1 128.3, 127.7, 127.4, 127.1, 127.0, 126.9. 126.0, 67.0, 47.5, 44.6, 40.7, 37.6, 36.3, 34.3; HRMS (FAB) 827.1775 (827.1776 calcd for C. _t2 H _2H Ν ₄ O ₀ F _? . MH).

N-9-fluorenylrnethoxycarbonyl-(/V ^* -t£?rt-butoxycarbonyl)- lysylglycylglycine (20). N-9-fluorenylmethoxycarbonyl-(N -/e/7-butoxycarbonyl)- lysine (0.9 g, 1.92 mmol) was dissolved, with N-hydroxysulfosuccinimide (458 mg, 2.1 1 mmol), in 5 mL 10% H ₂ 0/ DMF. l -Eihyl-3-[3-dimethylaminopropylJ- carbodiimide hydrochloride (EDC) (405 mg, 2.1 1 mmol) was added and the mixture was stirred for 4 h. The triethylammonium salt of diglycinc (492 mg, 2.1 1 mmol) was dissolved in 5 ml H ₂ O and added dropwise to the reaction mixture (The triethylammonium salt of diglycine was prepared as follows: Diglycine was dissolved in water and excess triethylamine was added. EtOH was added until the solution became homogeneous and the solution was evaporated to dryness in vacuo. The

resulting solid was suspended in MeOH, filtered, rinsed with Et ₂ 0 and dried in vacuo.) After stirring for 16 h the reaction mixture was partitioned between EtOAc and Na citrate buffer (0.2 M, pH 4.5). The aqueous phase was extracted with EtOAc (3 x 25 mL) and the combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated in vacuo. The resulting white foam was chromatographed on silica gel with 5% MeOH/CH ₂ Cl ₂ . Crystallization by the slow addition of pentane to a solution of the product in 10% EtOAc/CH ₂ Cl ₂ (25 mL) yielded 20 as a white powder (735 mg, 69%). Η NMR (300 MHz, DMSO-J ₆ ) d 8.17 (t, IH, 7 = 5.4 Hz), 8.09 (t, IH, 7 = 5.8 Hz) 7.87 (d, 2H, 7 = 7.4 Hz), 7.72 (dd, 2H, 7 = 7.2, 4.3 Hz), 7.53 (d, IH, 7 =7.8 Hz), 7.41 (t, 2H, 7 = 6.9 Hz), 7.32 (t, 2H, 7 = 7.0 Hz), 6.76 (t, IH, 7 = 5.3 Hz), 4.32-4.19 (m, 3H), 3.96 (br q, IH), 3.74 (t, 4H, 7 = 6.1 Hz), 2.88 (q, 2H, 7 = 4.5 Hz), 1.7- 1.1 (c, 6H), 1.35 (s, 9H); ^π C NMR (300 MHz, DMSO-</ ₆ ) d 172.3, 171.1, 169.1 , 156.1, 155.6, 143.9, 143.8, 140.7, 127.6, 127.1 , 125.3, 120.1, 77.3, 65.6. 54.7, 46.7, 40.6, 39.7, 31.4, 29.2, 28.3, 22.8; HRMS (FAB) m/z 583.2793 (583.2768 calcd for C ₃₀ H ₃ <)N ₄ O ₈ , MH).

N-9-fluorenylmethoxycarbonyl-(N ^< -t-'rt-butoxycarbonyl)- lysylglycylglycine pentafluorophenyl ester (21). Compound 20 (735 mg, 1.26 mmol) was dissolved in dry dioxane (6 mL) and pentafluorophenol (349 mg. 1.89 mmol), followed by dicyclohexylcarbodiimide (DCC, 285 mg, 1 .39 mmol) was added. After stirring for 5 h, the reaction was filtered, the precipitate was rinsed with dry dioxane (5 mL), and the solution was evaporated in vacuo. The resulting solid was triturated with hexanes, filtered, and dried in vacuo to yield 21 (803 mg, 1.07 mmol. 85%) as a white crystalline solid. Η ΝMR (300 MHz. CD ₂ C1 ₂ ) d 7.75 (d, 2H. 7 = 7.4 Hz), 7.58 (d, 2H, 7 = 7.4 Hz) 7.44 (br m, I H), 7.39 (t, 2H, 7 = 7.2 Hz). 7.28 (t. 2H, 7 =7.4 Hz), 7.06 (br m, IH), 5.96 (br m, IH), 4.81 (br m, I H), 4.38 (d, 2H, 7 = 7.1 Hz), 4.29 (d, 2H, 7 = 5.9 Hz), 4.19 (t, IH, 7 = 6.7 Hz), 4.05 (br t, I H). 3.97 (d, 2H, 7 = 5.6 Hz), 3.06 (br dt, 2H), 1.95- 1.3 (c, 6H) 1.30 (s, 9H); ' ⁵ C ΝMR (300 MHz, DMSO-J ₆ ) d 172.4, 169.8, 166.6, 156.1, 155.6, 143.9, 143.8, 140.7, 127.6, 127.0, 125.3, 120.0, 77.3, 65.7, 54.7, 46.7, 41.7, 32.9, 31.4, 29.2, 28.2, 22.8; HRMS (FAB) m/z 749.2579 (749.2610 calcd for C ₃₆ H ₃₈ Ν ₄ 0 ₈ F _s , MH).

Synthesis of Amino Acid Building Blocks

Statistical Mixture a R , = R ₂ = (CH ₂ )2CO ₂ Bn b R , =R ₂ = (CH ₂ ) ₂ NBOC c R1 =(CH 2> ₂ C0 ₂ Bn, Ft ₂ =(CH ₂ ) ₂ NBOC

O

two steps

Scheme 3 (a)

Synthesis of Amino Acid Building Blocks, cont'd

FMOCN

20

FMOCN

21

Scheme 3 (b)

EXAMPLE 4 SYNTHESIS OF AN AEDAMer COMPOUND

The AEDAMer compounds shown in Scheme 4 were synthesized as follows: As a prelude to the design of an appropriate AEDAMer backbone, an X-ray stmcture was obtained of the co-crystal between the monomers 3 and 4 (FIG. 3A and FIG. 3B). Taking this alignment of the rings as a probable energy minimum in solution, molecular mechanics simulations (MM2) predicted that the backbone shown in FIG. 3B would be compatible with the stacked stmcture of the co-crystal. Although any a-amino acid could be placed between the 1 ,5-dialkoxynaphthalene and 1.4,5,8- naphthalenetetracarboxylic diimide groups. Initially, (L)-aspartic acid residues were used for the backbone so as to increase water solubility and prevent aggregation. Compounds 14-16, as shown in scheme 4, were synthesized by solid phase methods.

General procedure for the solid phase synthesis of compounds 14, 15, and

16. Glycine-functionaiized 2-chlorotrityl polysyrene resin (Advanced Chemtech, 0.37 mmol/g loading, 200 mg = 0.074 mmol) was added to a 10 nil frittcd-glass filter flask equipped with a screw cap and teflon stopcock. The resin was shaken for a few minutes with 50% Et ₂ N/DMF (5 mL) and then rinsed with DMF (3 x 5 mL). /-PrOI I (3 x 5 mL), and DMF (3 x 5 mL). The resin was suspended in 2 mL DMF and 13 (210 mg, 0.27 mmol, 3.6 eq.) was added. HOBT (32 mg, 0.24 mmol, 3.2 eq.) was added and the mixture was shaken for 3 h using a simple oscillating shaker. (Completion of the coupling could not be monitored using the Kaiser test (Kaiser et ai, 1970) because compounds containing the 1 ,4,5,8-tetracarboxylic diimide moiety consistently gave a false positive reaction. The use of a 3 h coupling time and at least 2.5 equivalents of the pentafluorophenyl esters, however, generally gave high coupling yields based on analysis of the final products.) The resin was rinsed (DMF//- PrOH/DMF) and treated with 50% Et ₂ N/DMF for 30 min. The resin was rinsed again (DMF//-PrOH/DMF) and then 10 (210 mg, 0.30 mmol, 4.0 eq.) was added along with HOBT (32 mg). After shaking for 3 h, the resin was rinsed, depiotectcd, and rinsed again as described above, and the process was repeated until the desired number of

units had been coupled Alter the final deprotection step, the resin was treated with DIEA/Ac ₂ O/DMF (1 : 1 :4) for 30 min. to acetylate the N-terminus and rinsed Cleavage of the AEDAMers horn the resin was effected by tieatment with 5%> TFA/CH ₂ C1 ₂ for 10 mm. The resin was removed by filtration and the filtrate evaporated in vacuo. The /m-butyl esters were deprotected by treating the compounds with 1 : 1 TFA/CH ₂ C1 ₂ for 2 h Evaporation of the solutions in vacua yielded crude compounds 14, 15, and 16 as purple solids. These were evaporated from MeOH (2 x 10 mL), taken up in sodium phosphate buller (20 mM, pH 7.0), and filtered through a 0 45 m nylon syringe filtei. The solution was loaded onto a reversed phase C ₁₈ column (PepRPC 15m, Phaimacia) and eluted with a gradient ol 10 mM NH ₄ OAc/H ₂ 0→ CH^CN over 120 min with a flow rate of 0 75 mL/min. The main fraction was collected and determined to be homogeneous by HPLC (C _{i k} . same conditions as above). The product fiactions were pooled and lyophihzed to yield 14, 15, and 16 as purple solids, with yields in the lange ol 40-50% based on the initial loading of the resin

Solid Phase Peptide Synthesis of Aedamers

Resin Scheme 4 (a)

Solid Phase Peptide Synthesis of Aedamers, con't.

1 50% Et ₂ NH, DMF 30 mm 1 1 -1 3 HOAc/tπfluoroethanol/CH ₂ CI 2 ( cleavage from resin )

2 Repeat above steps n times 2 50% Trifluoroacetic acid/CH ₂ CI 2 ( deprotection of I- butyl esters

3 3 2 Acetic anhydπde/Et ₂ N, DMF 3 Purification by reversed phase FPLC (10 mM NH ₄ OAc — »- acetonitrile)

EXAMPLE 5 CHARACTERIZATION OF AN AEDAMer COMPOUND

The AEDAMer compound described in the previous example has been completely characterized using a variety of techniques and this example provide spectroscopic evidence dcmonstarting the folding of compounds 15 and 16 into a "pleated" structure in which the aromatic rings are stacked in a parallel, alternating fashion.

Aqueous solutions of compounds 14, 15, and 16 have a wine color due to broad charge-transfer absoφtion bands in the visible region. These bands showed linear Beer's Law behavior below 2 mM, indicating that observations made at or below these concentrations reflect only intramolecular interactions. The λ _ma χ(CT) value of the dimer 14 (522 nm) was identical to that of the complex between monomers 1 and 2(FIG. 2). A red shift of 18 nm was observed for the tetramer 15 and hexamer 16 (Scheme 4). Such a shift is consistent with ihe model for 15 and 16 in which more than two aromatic groups arc stacked simultaneously.

Significant hypochromism and shape differences were observed in the ultraviolet absorption bands of 14-16 when their UV/visible spectra were taken at identical chromophore concentrations. Oligomers consisting of totally non-interacting chromophores would have resulted in spectra equal in shape and intensity to that of a

1: 1 mixture of monomers at the same concentration. Hypochromism is distance- dependent as a function of 1/r and highly orientation-dependent with respect to the angle formed by the ring planes (Benniston et ai, 1994). Thus, the increasing hypochromism and shape differences that were observed in the scries 14-16 indicate that the aromatic chromophores were electronically coupled via a stacked or "pleated" arrangement in which the aromatic ring planes were parallel.

Fast atom bombardment (FAB) high-resolution mass spectra: calculated m/e for C ₄₈ H ₅ „N _y O| ₇ (5 as free acid, M ⁺ + H), 996.3263 and for C ₉₂ H ₉₂ N _{ι :} »O;,ι (6 as free

acid, M ⁺ + H), 1 ,874.6022. Found 996.328 1 and 1 ,874.6077, respectively. FAB low- resolution mass spectrum: calculated m/e for C Π ₆ H Π ₄ N | QO ₄₅ (7 as free acid, M ⁺ + H), 2,754.7. Found, 2,754.

500 Mhz Η NMR (20mM sodium phosphate buffer at pH 7 in D ₂ O) spectra are entirely consistent with the folding of structures 15 and 16 into well-defined pleated structures (Cantor & Schimmell, 1980). The chemical shift of the diimide hydrogens of uncomplexed 1 were taken as an approximation for that of a fully "unfolded" terminal diimide moiety, whereas the chemical shift of the fully complexed 1, as extrapolated from the NMR binding titration of 1 with 2, were taken as that of a "folded" terminal diimide moiety. The average chemical shift for the diimide hydrogens of dimer 14 lies between these two "cndpoints". indicating that under the conditions of the NMR experiment the dimer was only partially folded. On the other hand, the farthest downfield (least complexed) diimide signals in both 15 and 16 was slightly upfiekl from those of fully complexed 1. Thus, the carboxy- terminal acceptor groups were largely, if not entirely, stacked in 15 and 16, and their electronic environments were influenced by stacking of the internal aromatic groups. In addition, 2D COSY spectra revealed that several of the methylene hydrogens on the AEDAMer backbones were diastcreotopic, indicating a restriction of rotational motion on the NMR timescale. Finally, the expected nuclear Overhauser effect ( nOe) enhancements were observed between hydrogens of adjacent aromatic rings.

The above spectroscopic evidence demonstrates that the aedemer molecules 15 and 16 exhibit a well-defined pleated structure despite the inherent flexibility of the backbone. This, in turn, confirming the postulate that EDA interactions can serve as the basis for stabilizing novel higher order structural motifs.

EXAMPLE 6 SYNTHESIS OF A SERPENTERCALATOR

In order to facilitate polyintercalation, molecular mechanics calculations were used to model a flexible peptide backbone on a DNA template (Peek et ai, 1994). A four amino acid segment between diimide moieties was predicted by the computer simulation to have the optimum combination of length and flexibility, and one lysine residue was placed on each segment to provide electrostatic attraction to the DNA., The FMOC-protected pfp (pentafluorophenol) esters of synthetic amino acid building blocks, 19 and 21 (Scheme 3), were used to construct compounds 22-25. Compounds 22-25, shown in Scheme 5, were synthesized by standard solid phase peptide synthesis methods using the FMOC (9-fluorenylmethyloxycarbonyl ) strategy (Atherton & Shepard, 1989) with lysine-functionalized o-chlorotπtyl polystyrene resin (Barlos et ai, 1989).

General procedure for the solid phase synthesis of compounds 22, 23, 24, and 25. Lysine-functionalized 2-chlorotrityl polysyrene resin (Advanced Chemtech, 335 mg, 0.31 mmol/g loading) was added to a 10 ml fπtted-glass filter flask equipped with a screw cap and teflon stopcock. The resin was shaken Cor a few minutes with 50% Et ₂ N/DMF (5 mL) and then rinsed with DMF (3 x 5 mL), /-PrOH (3 x 5 mL), and DMF (3 x 5 mL). The resin was suspended in 2 mL DMF and 19 (212 mg, 0.26 mmol) was added. HOBT (35 mg, 0.26 mmol) was added and the mixture was shaken for 3 h using a simple oscillating shaker. (Completion ol the coupling could not be monitored using the Kaiser test (Kaiser et ai. 1970) because compounds containing the 1 ,4,5,8-tetracarboxylic diimide moiety consistently gave a false positive reaction. The use of a 3 h coupling time and 2.5 equivalents of the pentafluorophenyl esters, however, generally gave high coupling yields based on analysis of the final products.) The resin was rinsed (DMF//-PrOH/DMF) and treated with 50% EhN/DMF for 30 min. The resin was rinsed again (DMF//-P1OH/DMF) and then 21 ( 193 mg, 0.26 mmol) was added along with HOBT (35 mg, 0.26 mmol). After shaking for 3 h the resin was rinsed, deprotected, and rinsed again as described above, and the process

was repeated until the desired number of units had been coupled. After the final deprotection step the resin was treated with 1 : 1 :3 HOAc/2,2,2- trichlorocthanol/CH ₂ Cl ₂ (7 mL) for 1 h. The resin was filtered using a sintered glass funnel and the supernatant was evaporated to yield an olive green solid. To ensure that all of the compound had been cleaved, the resin was treated with 5% TFA/CH ₂ C1 ₂ for 10 min and filtered. The filtrate was evaporated in vacuo to yield an orange oil and combined with the original material that had been cleaved from the resin. To this mixture was added 1 : 1 TFA/CH ₂ C1 ₂ ( 10 mL) and, after 10 min, the solution was evaporated in vacuo. The resulting solid was evaporated from MeOH (2 x 10 mL), taken up in H ₂ 0 (20 mL), and filtered through a 0.45 m nylon syringe filter. The solution was loaded onto a reversed phase C _{I R} column (PepRPC 15m, Pharmacia) and eluted with a gradient of 10 mM TFA/fLO→ 0.8% TFA/CH,CN over 180 min with a flow rate of 0.75 mL/min. The main fraction was collected and determined to be homogeneous by HPLC (Cis, same conditions as above). The product fractions were pooled and lyophilized to yield 22, 23, 24, or 25 as a light green solid, with yields in the range of 25-35% based on the initial loading of the resin.

500 MHz IH NMR (D ₂ 0) spectra are entirely consistent with structures 22-25.

Compound 22. Η NMR (500 MHz, 10% D ₂ 0/H ₂ 0) d 8.64 (d, 2H, 7 = 7.6

Hz), 8.59 (d. 211, 7 = 7.6 Hz), 8.33 (t, IH, 7 = 5.8 Hz), 8.27 (d, I H, 7 = 7.5 Hz), 4.41 (m, 2H), 4.32 (t, 2H, 7 = 5.9 Hz), 4.23 (dt, IH, 7 =5.2, 7.9 Hz), 3.71 (s, 211). 3.64 (dt, 2H, 7 = 5.9. 5.8 Hz), 2.92 (br m, 2H), 2.72 (t, 2H, 7 = 6.8 Hz) 1 .72 (m, 2H), 1.61 (m, 2H), 1.34 (p, 2H, 7 = 7.6); HRMS (FAB) m/z 567.2222 (567.2203 calcd for C ₂₇ HιιN ₆ O _s . M).

Compound 23. Η NMR (500 MHz, D ₂ 0) tl 8.41 -8.32 (m. 8H), 4.48-4.29 (m,

5H). 4.25 (t. 2H, 7 = 8.9 Hz), 4.20 (t, 2H, 7 = 9.0 Hz), 4.1 1 -3.83 (m, 6H), 3.74 (s, 2H),

3.59 (m, 411). 3.03 (t, 2H, 7 = 7.7 Hz), 2.93 (t, 2H, 7 = 7.6 Hz), 2.86 (m, 2H), 2.71 (t, 2H, 7 = 7.1 Hz). 1 .94- 1.3 (m, 12H); HRMS (FAB ) m/z. 1229.4638 ( 1229.4652 calcd for Cκ9H| ₀ | N ₂₂ O ₂₆ , M-H).

Compound 24. Η NMR (500 MHz, D ₂ 0) d 8.43-8.22 (m, 12H), 4.32-4.1 1 (m, 15H), 3.99-3.82 (m, 12H), 3.77 (s, 2H), 3.68 (m, 6H), 3.01 (t, 4H. 7 = 7.5 Hz), 2.94 (t, 2H, 7 = 7.6 Hz), 2.77 (m, 4H), 2.66 (t, 211, 7 = 7.2 Hz), 1.90- 1 .29 (m, 18H); HRMS (FAB) m/z 1891.7175 (1891.7101 calcd for Cs ₉ H| ₀ ιN ₂₂ O ₂₆ , M-2H).

Compound 25. 1H NMR (500 MHz, D ₂ 0) d 8.4-8.1 (m, 16H). 4.38-4.1 (m, 20H), 3.98-3.70 (m, 18H), 3.77 (s, 2H), 3.55 (m, 8H), 3.02 (t, 6H, 7 = 7.5 Hz), 2.96 (t, 2H. 7 = 7.5 Hz), 2.75 (m, 6H), 2.66 (t, 2H, 7 = 7.3 Hz), 1.90- 1.3 (m, 24H); MS (FAB) m/z. 2554 (2554 calcd for C, ₂₀ Hι ₃₆ N ₃₀ O ₃₅ , M-4H). Fast atom bombardment (FAB) high resolution mass spectra: calculated m/e for C ₂₇ H ₃ |N _< sOκ (22 as free base, M+ + 2H). 567.2203, for C ₅ κ H _ή 6N| ₄ 0| ₇ (23 as free base, M+ + 2H), 1229.4652 and for C _S oHιoιN ₂₂ 0 ₂₆ (24 as free base, M+ + 2H), 1891.7101. Found. 567.2222, 1229.4638 and 1891.7175, respectively. FAB low resolution spectrum: calculated for Ci2oHi ₃ fiN3oO ₃ 5 (25 as free base, M+ + 1 ), 2554. Found, 2554.

Solid Phase Peptide Synthesis of Polyintercalators

Scheme 5 (a)

Solid Phase Peptide Synthesis of Polyintercalators, con't

1 113 HOAc/tnfluoroethanol/CH gCI 2 ( cleavage from resin }

1 50% Et ₂ NH, DMF, 30 mm 2 50% Trifluoroacetic acid/CH 2 CI 2 ( deprotection of BOC groups

3 Purification by reversed phase FPLC

2 Repeat above steps n times

(10mMTFA/H ₂ 0 •- 08% TFA/ acetonitrile)

22 n = 0 23 π=1 24 π =2 25 n=3

Scheme 5 (b)

EXAMPLE 7 CHARACTERIZATION OF DNA BINDING

Compounds 22-25 exhibited significant hypochromism in their ultraviolet absoφtion bands upon mixing with excess calf thymus DNA (Table 4). Intercalating species are known to exhibit hypochromism when bound to DNA due to the stacking interactions present in the bound complex (Wakelin, 1986). Importantly, the extinction coefficients for compounds 23-25 when bound to DNA were equal to the number of diimide units present in the molecules multiplied by the extinction coefficient observed for bound compound 22 (Table 4). This additivity of the extinction coefficients per diimide group for the compounds bound to DNA was consistent with all the diimide groups being intercalated simultaneously for each molecule. Any diimide groups remaining free in solution would be expected to contribute a larger absorbance and thus increase the overall value above that observed. In the absence of DNA, the larger molecules exhibit extinction coefficients that are smaller than multiples of that seen with unbound compound 22. This was interpreted as providing evidence that when not bound to DNA. the larger molecules exhibit some degree of self-stacking between the diimide groups.

TABLE 4. EXTINCTION COEFFICIENTS BOUND TO DNA

COMPOUND ε (free)" ε (bound) ¹ φ (degrees) ^*1 m ^c n ¹

22 20,000 10,400 12 .41 2

23 27,400 20, 100 24 .84 4

24 39,200 27,700 37 1.4 6

25 51 ,300 39,500 48 1 .9 8

Viscometric studies were carried out in order to ascertain further whether all of the aromatic groups in each compound were simultaneously intercalated upon binding to double-stranded DNA. The helix extension parameter, which is a measure of the lengthening of linear fragments of double stranded calf thymus DNA, was determined

for each compound by the method of Cohen and Eisenbcrg ( 1969). The helix extension parameter equals the slope ol the line given by the equation:

L/L0 =l+mr

where L/LO is the cube root of the reduced viscosity and r is the ratio of bound compound per DNA base pair. As shown graphically in FIG. 5, the slopes increase in increments according to the number of diimide groups per molecule. This data indicates that the molecules are indeed fully intetcalated. Moreover, the fact that the slopes arc additive shows that the peptide tether has little effect on the degree to which each intercalating moiety lengthens double-stranded DNA. The ratio of DNA base pairs per bound intercalatoi, n, was determined as the point at which the viscosity of linear DNA no longei increased upon addition ol more compound Fiom these experiments, values of n equal to 4, 6 and 8 were detei mined with compounds 23-25, respectively. This data is in agreement with the neaiest-neighbor exclusion principle (Ciotheis, 1968) in which each intercalated unit is separated by two base pairs. To obtain n lor the monomer 22, the binding isotherm between 22 and CT DNA was determined spectrophotometπcally and fit to the McGee-von Hippel equation (McGhee & von Hippel, 1974), which defines both n and the equilibrium constant (Ka) between molecules with multiple contiguous binding sites (MG 15) As expected, the value n=2 was derived for the monomer.

The unwinding angles for compounds 22-25 weie determined by the method of Revet, et al., ( 1971 ) using viscometπc titrations with closed circular supercoiled plasmid (CCS) DNA (FIG. 6). As with the helix extension parameters, the unwinding angles foi 23-25 were multiples of that found foi 22. In addition, the unwinding angle value determined for 22 falls within the range observed lor othei monointercalators utilizing the diimide gioup (Yen et ai. 1982). Together with the observed additive hypochromism and lengthening data, these unwinding angle measurements demonstrate that all of the diimide groups were simultaneously intercalated when 22- 25 bound to double-stranded DNA (FIG 14).

In the case of the tetramer compound 25, all foui ol the aromatic diimide groups were simultaneously intercalated. Taken with the bound extinction coelficients, this demonstrates tetraintercalation Despite at least one published attempt (Wirth, 1988), this represents the first example ot tetraintercalation by a single molecule.

Two possible modes of intercalation are contemplated foi the polyintercalators of the present invention as shown in FIG 4A and FIG 4B FIG. 4A shows a stmcture in which the aromatic groups are stitched through the double helix such that their linker segments reside in the major and minor grooves in an alternating fashion Alternatively shown in FIG. 4B is a structure in which the diimide gioups intercalate into double-stranded DNA, while the linker segments leside entirely within the ma)oι groove.

EXAMPLE 8

ALTERNATE LINKAGE GEOMETRIES

Many new backbone linkage geometries aie contemplated toi use with the present invention Seveial examples ot which aie outlined in the piesent example

Hairpins and Knots

The original AEDAMers exhibit a "pleated" stiucture in aqueous solution at temperatures up to 70° C by vπtue of the alternating sequence ol aromatic donor and acceptoi lesidues linked togethei; for example, the AEDAMer having the sequence C- A-Asp-D-Asp-A-Asp-D-Asp-A-Asp-D-Asp-NAc. Here, "C and "NAc" aie the carboxyl terminus and acetylated amino terminus, respectively; "A" and "D" are the diimide acceptor and naphthalene donor units, respectively; and "Asp" is L-aspartic acid. This abbieviated notation will be used throughout the iest ot the example

A different type of AEDAMer has been synthesized, with the non-alternating sequence C-A-Asp-D-Asp-D-Asp-A-Asp-NAc. In aqueous solution, this molecule also exhibited the hypochromism, red-shifted chargc-transici band and NMR spectra that arc consistent with a fully stacked structure. This structure has been designed to form a structure in which the last A residue folds back and inserts between the two D residues, somewhat analogous to a nucleic acid haiφin. The same linkage chain used for the pleated system can also accommodate the hairpin structure, because the distance that must be traversed by linkages m both systems is nearly the same (-6.6 A) In the normal pleated structure the linker must accommodate a 1/4 turn along with the shorter lineai distance between adjacent residues, while in the hairpin, the linkage must travel a greatei lineai distance between non-ad|acent icsidues, but no turn is lequired and the distances balance out

Designs arc also contemplated foi new linkage geometries of an AEDAMer "haiipin", with the sequence C-A-Asp-A-Asp-A-Asp D-Asp-D-Asp-D-Asp-NAc and an AEDAMer "knot", with the sequence C-D-Asp-A-Asp-A-A>p-D-Asp-D-Asp-A-

Asp-NAc. These molecules should adopt the desncd structuies in ordei to allow foi the greatest possible stacking ol the D and A residues

B. AEDAMers In Organic Solvent

AEDAMer structures aie also contemplated loi use with organic solvent. Because the stacking interaction between the election-deficient and electron-rich monomer units is largely electrostatic in natuie, these structuies should be largely solvent independent.

Hydrophobic AEDAMers, for example, (C-A-Leu-D-Leu-A-Leu-D-Leu-A-

Leu-D-Leu-NAc), will contain L-leucine residues instead of the L-aspartic acid residues found in the original AEDAMer. The C-tei minus will be produced as the primary amide. The spectroscopic techniques desciibed above allow for the study of

AEDAMer structure in a variety of organic solvents such as DMSO. DMF, diethyl ether, CHC1 ₃ and hexane

C. Zipper or Bundle Structures

Amphiphihc α-hehces composed of amino acids pack into largei assemblies including the so-called "leucine zippers" formed between two helices, and the so- called "helix bundles" formed from the association of four helices In both cases, the hydrophobic amino acid side chains associate with each other, while the hydrophilic side chains face out into the aqueous solution

This can be applied to construct amphiphihc AEDAMei structuies Foi example, the construction of an AEDAMer that has hydrophobic L-leucine residues on one side and hydrophilic L-aspaitate residues on the othei would be expected to form such a structure. These AEDAMer^ will self-associate in aqueous solution, in well-delined ways The associated AEDAMers will be analyzed by size-exclusion HPLC, and the retention times of the AEDAMer assemblies will be compared to standards ol known size in order to determine the predominant species present

This will allow toi AEDAMers constructed with covalent links between the

AEDAMer domains Foi example, if the AEDAMers pielei to adopt a two-domain "zipper" type of structure, then two AEDAMers domains will be linked together by a flexible chain Similaily, if a three or four helix bundle is piclerred, then three or four AEDAMer domains will be linked together, iespectively In this way, AEDAMer stiuctures appioaching the size of natuial proteins will be pioduced

D. An AEDAMer That Inserts Into Lipid Bilayers

An AEDAMei construct is contemplated for inseition into a lipid bilayer This AEDAMer, for example, may be composed of an alternating aπangement of foui diimide and toui naphthalene units, linked by hydrophobic L-leucine iesidues Theic

will be positively charged groups on each end of the structure, in the form of an L- lysine residue on the C-termmus, and a free amino group as the N-termmus. The carboxyl terminus will be blocked as the primary amide This AEDAMer is the right size (-30 A in length) and should posses the correct hydrophobic/charge properties to insert into a phosphohpid bilayer.

The AEDAMer-phosphohpid bilayer will be analyzed by examining the UV- vis and NMR spectra of the AEDAMer in the presence and absence of phosphatidyhnositol liposomes. The largely hydrophobic AEDAMer will likely be insoluble in aqueous solution in the absence of liposomes, but in their presence, the familuu chaige-transfer band and chemical shifts will be evident if the AEDAMei becomes stacked inside the lipid bilayei.

E. Electron Transfer Studies Using AEDAMers With Arrays Of Metal Complexes

The AEDAMer scaffold presents opportunities lor positioning arrays of functional moieties, including redox active metal complexes, at delined onentations with respect to each other. For example, the study of election tiansfer between a kinetically inert, photoexcitable electron-donating metal species such as Ru(NH ₃ )«; ²⁴ , (Winklery & Giay, 1992) and a kinetically men, election acceptor species such as an Fe ⁺ -poφhyπn complex may be conducted. Standard time-iesolved can be used to measure the electron transfer rates (Shreder et al, 1995) In these AEDAMers, the ¥c ^λ+ -porphyπn moiety can be moved systematically farthei away from the Ru(NH,) _s ²⁺ unit Each AEDAMer will have one Ru(NH,V" and only one Fe ¹⁺ - poφhyπn attached, but the distance between the Ru(NH^ ² ' and Fc ^,+ -porphyπn moieties can be systematically increased along the same and opposite faces of the AEDAMers

EXAMPLE 9 AEDAMer Linker Libraries-Optimization of Folding:

Synthesis Libraries can be synthesized in which the amino acid linkers between the donor and acceptor groups of AEDAMers are randomized via a split synthesis. The same FMOC-based synthetic procedures used to make the original AEDAMers can be used. Following deprotection of the FMOC group of the preceding residue, the beads will be split into different reaction vessels and reacted with different residues in the presence of a coupling agent, for example, HBTV. Following coupling, all the beads are collected in the same vessel, the terminal FMOC group removed, the sample split again, and the process continued. In this way, the amino acid linker will be randomized, but the AEDAMer on any one bead will have the same sequence. Both the number and the type of residues in the linker will be varied.

Analysis Automated protein sequencing techniques on single beads will be used to determine the sequence of any member of the library. In addition, amino acid analysis can be used to analyze the composition of amino acids on any one bead. Finally, mass spectral techniques may be used for the sequence determination. (Stork et al., ).

Library Screens The AEDAMer linker libraries will be screened for folding by exploiting the visible region charge-transfer band that appears when an AEDAMer folds correctly. Therefore, any wine-colored beads in the library will be folded, and these can be removed and the linker sequence determined. In this way, linkers may be optimized with respect to length and residue composition. By screening the libraries for the wine color under different solvent, temperature, buffer conditions, linkers, etc., that are stable to these various conditions can be found.

EXAMPLE 10 Kinetic StudiesJDNAse I and Chemical Footprinting

Kinetics Studies To investigate the dynamics of the interactions of 22-25 with DNA, association and dissociation rates were determined for CT DNA, poly(dAdT) and poly(dGdC) (Tables 5 and 6, respectively). The association rates and monomer dissociation rates required stopped-flow techniques, while the slower off- rates for compounds 23-25 were measured using a standard spectrophotometcr. All dissociation rate measurements required the presence of 2% SDS in the buffer to sequester the dissociated intercalators. Control studies revealed that compounds 22- 25 did not reassociate with the nucleic acids in 2 ^f /< SDS, but at lower SDS concentrations, reassociation was observed.

Table 5. Association rates

22 ^a 23 24 25

DNA type k ^h (s- A ^c (%) kV) A ^c k ( S ) A ^c (%) k ^b (s-' ) A ^{c ]} ) (%) (%)

12.0 100 40. 1 33 41 .9 38 28.4 32 poly(dAdt) 4.1 48 4.9 44 4.8 42

0.90 19 0.99 18 1.2 26

29.2 22 6.6 37 5.7 40 4.7 49 poly(dGdC) 8.3 78 1.3 25 0.70 38 0.60 27

0.28 38 0.083 22 0.094 24

38.5 42 3.9 36 9.6 23 8.4 29

CT DNA 9.7 43 1.2 31 2. 1 41 1.6 38

0.78 15 0.25 43 0.28 36 0.28 33

^a Numbers refer to compounds 22-25. ^b Association and dissociation rate constants determined by fitting the spectrophotometrically-determined decay traces to up to three exponentials. ^c Relative amplitudes from curve fits as percentage of total.

Table 6. Dissociation rates

22 23 24 25

DNA type k ^b (s') A ^L (%) k ^b (V) A ^c (%) k ^b ts ^! ) A ^c (%) k ^b (s') A ^c (%)

154 27 012 8 0097 12 Oil 7 poly(dAdt) 66 73 0021 92 0021 88 0019 93

098 23 59x 10 ⁴ 8 69x 10 ⁴ 6 46x 10 ⁴ 5 poly(dGdC) 0047 77 46x 10 ⁵ 15 15 37x 10 ⁵ 15 38x 10 ⁶ 77 79 23x 10 ⁶ 80

51 24 55x 10 ³ 34 60x 10 ^s 37 43x 10 ³ 39

CTDNA 10 36 50x 10 ⁴ 35 55x 10 ⁴ 35 50x 10 ⁴ 46

023 40 lOx 10 ⁴ 31 11 x 1O ⁴ 28 8 lx 10 ⁵ 15 o ^a Numbeιs lefer to compounds 22-25 ^b Association and dissociation rate constants determined by fitting the spectiophotometncally-determiπed decay tiaces to up to three exponentials

^L Relative amplitudes fiom cuive fits as peicentage of total

For all of the association profiles, calculated fits represent the minimum number of exponentials required to give purely random residuals. Most of the dissociation profiles were also adequately described by up to three exponentials Dissociations of compounds 23-25 from CT DNA, however, were too complex to be fit even to a tπ-exponential decay process, and reproducibly non-random residuals were obtained in each of these cases. This complexity is not unexpected considering the sequence heterogeneity of natural DNA and the large number of potential binding sites. Compound 1, with only one diimide unit, shows distinct kinetic behavior that is generally taster, both in terms of on- and of f- rates, and less complex than the polyintercalators 23-25.

The kinetic data reveal that 22-25 exhibit a diamatic preference foi poly(dGdC) over poly(dAdT). reflected pπmai ily in their dissociation rates While the dissociations of compounds 23-25 from poly(dAdT) are essentially complete within 200 s, the longest (and most piedominant) component of their dissociations fiom poly(dGdC) has a half-life of at least 48 h. Interestingly, compounds 23 25 have virtually identical dissociation rate piofiles, in terms of their time constants and ielative amplitudes The slowest components for the dissociation ol 23-25 from poly(dGdC) could not be determined with accuiacy because the samples degraded over the long period required to observe complete decay of the complex Experiments weie peiformed in which compounds were mixed with poly(dGdC) dissolved in 2% SDS, and in no case could reassociation be obseived, even ovei a period of several days. This indicates that the dissociations from poly(dGdC) go to completion and not to some partially bound equilibrium state To fit the dissociation cuivcs for poly(dGdC), the asymptote representing complete dissociation was set as a constant according to the known extinction coefficients of the compounds in 2% SDS.

DNAse I Footprinting. A synthetic ohgonucleotide duplex was used for the footpπnting studies that contained three GC sites of 4, 6. and 8 base pairs in length, separated by stretches of AT. The footpπnting gels (FIGS 7 and 8) leveal the

predicted preferential binding at the GC tracts for all the molecules including the monomer 22. For each of the polyintercalators 23-25, all three of the GC sites became occupied simultaneously within a narrow range of polyintercalator concentration. The dimer and tetramer show identical footprinting patterns; the only difference between corresponding lanes is that a two-fold higher concentration of 23 is required to produce the same footprint made by 25. Although the footprint of the trimer is similar to those of the dimer and tetramer, there is an important difference: Whereas the dimer apparently binds cooperatively to the GC sites, the footprint of the trimer appears to increase gradually in size as a function of concentration. For example, the trimer displays a significantly smaller footprint at 125 nM than at 250 nM, while the dimer appears to be completely unbound at 125 nM and completely bound to the GC sites at 250 nM.

Chemical Footprinting. Chemical footprinting methods are often employed to elucidate the groove and sequence preferences of DNA-binding molecules. Using EDTA, which cleaves DNA largely via the minor groove, and dimethyl sulfate (DMS), which alkylates the N7 position of guanine and causes cleavage via the major groove, footprinting experiments were carried out with the same oligonucleotides used in the DNAse 1 studies. Studies were performed under conditions in which 22-25 arc known to be bound, but in no case was protection from cleavage by either chemical reagent observed. On the contrary, in the DMS experiments enhancement of methylation-induced cleavage was seen at most of the bound guanosine positions, possibly indicative of distortion at the bound GC sequences.

The similarity in the rates of association of the four compounds with a given type of DNA can be related to a rate-determining association step that is independent of the structure of the ligand. Indeed, such similarity in on-ratcs was observed among a series of mono-, bis-, and trisacridines with poly(dAdT) and was attributed to rate- limiting desolvation of both the ligand and the DNA to form a pre-association complex (Laugaa et al., 1985; Capelle et al., 1979). Since polyintercalators 23-25 exhibit a significant degree of self-stacking in aqueous solution, perhaps instead of

-71 desolvation a rate-limiting self-d/y sociatwn step is required before these molecules are able to intercalate This is consistent with the observation that the monomei which is not significantly aggregated under the conditions of the association experiments (vide supra), displays significantly faster association rates than 23-25 lor the different DNAs

Cooperativity. A synthetic ohgonucleotide duplex was used toi the footprinting studies that contained three GC sites of 4, 6, and 8 basepairs in length, separated by stietches ol AT The footprinting gels (FIGS 7 and 8) leveal the predicted pieferential binding at the GC tracts toi all the molecules including the monomer 22 For each of the polyintercalatois 23-25, all thiee ot the GC sites became occupied simultaneously within a very nanow range ol polyinteicalator concentration The dimer and tetiamei show identical lootpπnting patterns, the only difleience between conesponding lanes is that a two-lold higher tonccntiation of 23 is lequned to pioduce the same footpiint made by 25 Impoitantly, at no concenliation is any footpiint smallei than 9 basepairs obsei ved with 23 (oi 25) I hat is binding of two molecules of 23 simultaneously is more favorable than binding ol a single molecule of 23, thereby explaining the 9 basepan footprint e\ en at the 4 basepair GC site

Binding modes. The DNA-bound state of a tetiainteicalaling polydnmide such as 25 can be repiesented by at least two distinct schematic models (FIG 9) Yen, et al obseived the inteicalation of seveial compounds containing the 1 ,4,5,8- naphthalenetetiacaiboxyhc diimide moiety with bulky gioups such as adamantyl attached at both of the nitiogen atoms (Yen ei al , 1982) ^r l hesc authois pioposed that in ordei to inteicalate at least one of the bulky gioups must pass thiough the double helix, invoking a dynamic model of DNA that allows foi tiansient unwinding, even at tempeiatures well below its melting tcmperatuic This type of "threading ' intercalation has been attributed to other DNA binding compounds, namely the drug nogalamycin (Fox and Wanng, 1984, Williams et al 1990), and Takenaka et al (1993) repotted a fm-intercalating molecule that was proposed to bind in a thieading mannei Applying this reasoning to compounds 24 and 25 of this system lesults in a

structure, referred to here as model A, in which the aromatic groups thread through the double helix such that their linker segments reside in the major and minor grooves in an alternating fashion (FIG. 9A).

An alternative possibility for the bound structure, referred to here as model B, is one in which the diimide groups intercalate into double-stranded DNA "edge-on", while the linker segments reside entirely within the major groove (FIG. 9B). The helical twist of the DNA duplex could allow for considerable intercalation of the diimides, while still providing enough space for the linker chains to extend from each end of the diimide. Of course, some hybrid of these two modes is possible with 24 and 25. In the absence of definitive structural data from either NMR spectroscopy or X-ray crystallography (these methods have yielded little information to date), the inventorshave relied on more indirect routes to discern the binding modes of compounds 22-25. Based primarily on kinetic evidence, the inventorsconclude thai of the two model B is more consistent with the available data.

All components of the stopped-flow association kinetics of 22-25 with poly(dGdC) are several times slower than those for poly(dAdT), implying that transient unwinding of the double helix (an event that is about 25 times more likely for an AT than a GC base pair)-' ⁴ - ³⁵ has an influence on the rates of association. This would tend to favor model A; indeed, Fox and Waring observe a similar trend in the association of the threading intercalator nogalamycin (Fox and Waring, 1984), certain components of which they ascribe to a rate-determining DNA breathing step. If, however, model A is correct and DNA breathing is rate-determining, then one would not expect similar kinetic behavior among all of the polyintercalating species. Relative to the bis-intercalator 23, the tris- and tetra- intercalators would require extra, presumably rate-limiting, steps to thread into and out of the double helix. On the other hand, DNA breathing might facilitate intercalation by making the major groove more sterically accessible, consistent with binding via model B. Instead of rendering the association kinetics more complex for the longer intercalators, this effect would be similar for all of the polyintercalators (thus giving rise to similar rate profiles).

Moreover, according to model A one would expect 24 and 25 to display significantly slower off-rates compared with 23 A salient feature of the dissociation data is that, within a given type of DNA, compounds 23-25 display remarkably similar kinetic behavior This includes all of the key parameters of the fits, including the number of components observed, the relative extent of each component, and the magnitudes of the derived constants. No reasonable mechanistic model that involves "unthreading" of either of the longer intercalators 24 or 25 could be produced that fits the dissociation data On one hand, model A cannot be rigorously ruled out For example, it is possible, albeit unlikely, that the "threading" steps do not produce spectroscopic changes that are detectable by the absorbance techniques used in these studies On the other hand, the mechanism below for the dissociations of 23-25 from poly(dAdT) is provided that is consistent with all of the available data only if the compounds are bound according to model B

EXAMPLE 11

Mechanism of Dissociation.

This example investigates whether a mechanistic scheme within model B could be generated that predicts the similarity in dissociation profiles between the different compounds for a given type of DNA The inventors began with the assumptions that (a) the elementary steps in a sequential mechanism should be similar among all three of the polyintercalators 23-25, (b) the N-to C-terminal asymmetry of the compounds could be ignored and (c) that in the presence of SDS, which solvates the unbound chromophores, bi-molecular re-association rates are negligible These assumptions allowed the inventors to prune the many branching steps that are theoretically possible for a multi-intercalating species down to a manageable number The potential mechanisms were then evaluated by comparing the resulting theoretical dissociation curves with the actual data

Specifically, theoretical absorbance-vs -time curves were constructed assuming a constant increase in absorbance per chromophore expelled from the DNA A rate

equation for the unbound species was generated for a given mechanism and substituted into the absorbance expression. The resulting differential equation was numerically solved for a given set of rate constants and plotted against the experimental data. The rate constants were manually varied and the resulting plots were used to obtain an interpolative fit.

Initially it was attempted to reproduce the dissociation profiles of all three compounds with a simple mechanistic scheme in which the intercalating groups dissociate in no particular order and at approximately the same rate. This model was unable to produce satisfactory fits for any set of experimental data.

In an alternative approach, the inventors introduced as a constraint the fact that the dimer 23 binds cooperatively in units of two (as observed in the DNAse I footprinting experiments.) In such a scenario, the rates of dissociation of the individual intercalating groups are largely dependent upon the occupancy of neighboring sites. For the sake of simplicity, the inventors postulated a step-wise non-branching mechanism (FIG. 10), based on the following assumptions: First, since cooperativity was observed with the dimer, it was inferred that binding to the third, but not necessarily to the fourth occupied site is cooperative for each of the compounds. Second, the fact that the K _cq for the dimer is less than the square of the K _eq of the monomer implied that the binding of adjacent groups (separated by two base pairs) is non-, or perhaps even anti-cooperative. In terms of off-rates this data suggested the order k _lh i _r d < k _fιrs , A k _sc∞nd A kf _our , _h for the dissociation from the respective intercalated sites.

FIG. 13 A-C show the best fits obtained for the dissociation of compounds 2-4 from poly(dAdT), according to the present model (FIG. 10). The resulting rate constants are listed in Table 7 (note the similarity in the rate constant sets for the three compounds). Flowevcr, the postulated mechanistic scheme failed to produce satisfactory fits for the poly(dGdC) data.

The results shown in Table 7 for the poly(dAdT) data support the initial assumptions Numbers in parentheses indicate "flexible" rate constants whose values could be varied significantly (within an order of magnitude) without a large impact on the fit. These numbers show k ₃ to be the rate limiting step, and attempts to reverse the relative size of the constants failed. Note that for the tπmer and tetramer the proposed order of dissociation from the intercalated sites would require the existence of intermediates in which dissociated chromophores are tethered on both sides by intercalated groups (FIG 10). However, given the small diffeience between k ₂ and k ₃ , it is possible that any "middle" bound residue in the turner and tetramer would held tightly in position by the linkers on both sides, giving rise to an alternative mode of dissociation (FIG. 1 1, pathway b).

Table 7.

Rate constants obtained from fitting dissociation eui vcs ol compounds 23-25 with poly(dAdt) according to the mechanism in scheme 1.

COMPOUND 23 24 25 k," 0.12 0.1 1 k_, 7 x 10 ⁴ 5 x 10 ⁴ k ₂ 7 x 10 - 0.12 7.0 x 10 ² k.2 (5 x ιo-y (5 x 10 ⁴ ) (5 x 10 ^"4 ) k ₃ 2.1 x 10 ^"2 2 1 x 10 - 2 1 x 10 ² k ₃ (5 x 10 ⁴ ) (5 x 10 ⁴ )

magnitude without greatly affecting the closeness of fit

This mathematical model does nol implicitly differentiate between intermediates such as a and b in the dissociation teaction of compound 24, as the absorbance curve is simulated by simply counting the number of unbound chromophores in each species. For the dimer 23. wheie the two corresponding intermediates (FIG 9) theoretically give rise to kinetically distinct pathways, the inventors observed an equally good fit for both mechanisms (via intermediates c oi d, corresponding to a and b for the trimer, respectively), with the same set of rate

constants. This is not surprising considering the negligible reverse rate constants (k. _2a

A k. _3h A 0.0005 s ^" ) in both instances. In light of the above, the inventors propose that in reality the dissociation proceeds through a combination of these two pathways. Since each elementary step is virtually irreversible in the poly(dAdT) fits, the constants obtained are possibly linear combinations of the elementary step rate constants.

The existence of such alternate pathways may account for the more complex dissociation curves observed for poly(dGdC). The slower experimental off-rates measured for all three compounds, in addition, suggest both smaller dissociation and larger reverse (re-association) constants, which may lead to more complex kinetic behavior, than for poly(dAdT). Furthermore, one or more of the assumptions that allowed a fit of the poly(dAdT) data may not be valid in the case of the dissociations from poly(dGdC). For example, a branching of the mechanistic steps would have to be introduced in order to take into account the N- to C-terminal asymmetry of the polyintercalators, a factor which may be important on the much slower timescale of the poly(dGdC) dissociations. In order to take this branching into account, many more kinetic constants would have to be introduced, which seriously limits the credibility of any physical model deduced from a manual fit.

The inventors have described a new class ol polyintercalating compounds, and the first known tetraintercalator. Using DNAse I footprinting, the inventors found that cooperativity is an important feature in the binding of the present polyintercalators to double-stranded DNA. Using kinelic data obtained from stoppcd-flow and manual mixing techniques, allowed the proposal of a mode of binding for compounds 23-25 and a mechanism for their dissociations from poly(dAdT). The solid phase synthesis of these compounds, in which the intercalating groups are attached to one another by peptide linkers, suggests the possibility of developing novel DNA-binding compounds using the rapidly advancing techniques of combinatorial and solid phase chemistry. With this design the inventors may employ non-peptide solid-phase synthesis to

generate polyintercalators separated by rigid, highly functionalizcd linkers in which properties such as sequence specificity or enzyme inhibition (i.e., topoisomerase inhibition) could be selected from a library or rationally designed.

EXAMPLE 12

Polyacceptor Libraries-Isolation of Compounds with Desirable Intercalation

Properties:

Synthesis Libraries will be synthesized in which the amino acid linkers of the polyacceptors are randomized via a split synthesis. For example, the same FMOC- based synthetic procedures used to make the original polyacceptors can be used. Here, following deprotection of the FMOC group of the preceding residue, the beads will be split into different reaction vessels and reacted with different residues in the presence of a coupling agent, for example, HBTV. Following coupling, all the beads are collected in the same vessel, the terminal FMOC group removed, the sample split again, the process continued. In this way, the amino acid linker will be randomized, but the polyacceptors on any one bead will have the same sequence.

Analysis Automated protein sequencing techniques on single beads will be used to determine the sequence of any member of the library. In addition, amino acid analysis can be used to analyze the composition of amino acids on any one bead.

Finally, mass spectral techniques may be used for the sequence determination. (Stork et al. )

Library Screens 1 ) Fluorescently-labelcd nucleic acids can be incubated with the library, so that beads containing high affinity molecules will become fluorescently labeled. In this way, the best DNA-binding molecules can be quickly identified out of the library. 2) Libraries will be fractionated on the basis of physical properties; then the different fractions will be screened for cytotoxicity or other biological activity. In order to accomplish this, the polyintercalating molecules will be cleaved from the solid support, and then subjected to preparative chromatography on columns that contain C18, C8, and C4 reverse phase material, silica gel normal

phase material, catiomc and anionic exchange materials, etc Fractions from each type of chromatography will be screened for biological activity and the most active fractions will be analyzed to determine their composition via amino acid analysis In this way, any correlation between gross chemical properties and biological activity will be identified. The composition information from the active fractions will be used to cieate a next generation library enriched in the identified lesiducs The process will be repeated several times in an effort to identify important molecular property- biological activity relationships, and at the same time iteiatively deduce optimized drug candidates.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light ot the present disclosure While the compositions and methods of this invention have been described in terms ot preterred embodiments, it will be apparent to those of skill in the art that vanations may be applied to the compositions and/oi methods and in the steps or in the sequence of steps of the method described herein without depaiting from the concept, spirit and scope of the invention. More specifically, it will be apparent that ceitain agents which aie both chemically and physiologically i elated may be substituted for the agents described herein while the same or similar results would be achieved. All such similai substitutes and modifications apparent to those skilled in the art aie deemed to be within the spirit, scope and concept of the invention as delined by the appended claims

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference.

Amabilino.6 ^> t π/. , J Am. Chem. Soc , 1 17: 1271 - 1294. 1995.

Ashton et ai, J. Chem. Soc, Chem Commun., 1677- 1679, 1991 .

Atherton & Shepard. Solid Phase Peptide Synthesis. IRL. Oxford, 1989.

Atwell et al, J. Am. Chem. Soc, 105: 1913- 1914, 1983.

Barlos et ai , Tetrahedron Lett. , 30:3943-3946, 1989.

Benniston et ai, Tetrahedron Lett., 35: 1473- 1476, 1994.

Bruce et ai, Rapid Communications in Mass Spectrometry, 9:644-650. 1995.

Burbaum et ai, Proc Natl. Acad. Scie USA, 92:6027-603 1 , 1995.

Cantor & Schimmel. Biophysical Chemistry, Part II, Freeman, New York, 390-408, 1980.

Capelle et al., Biochemistry 17: 3354-3366, 1979.

Chambron et ai, in: Transition Metals in Supramolecular Chemistry, Fabbrizzi & Poggi (eds), 371-390, Kluwer Academic, Dordrecht, 1994.

Cohen, G.; Eisenberg, H. Biopolymers, #45-49, 1969.

Crothers, Biopolymers, 6:575, 1968.

Delepiene, et al., Biopolymers , 31, 331 -353, 1991.

Denny et ai , Biophys. Chem., 22: 17-26, 1985.

Fox and Waring, Biochim. Biophys. Acta, 802: 162- 168, 1984.

Freicr et ai, J. Med. Chem, 38:344-352, 1995.

Hansen, et al., Biochemistry , 22: 4878-4886, 1983.

Herbstein, in Perspectives in Structural Chemistry, Vol.4, Divinity & Ibers (eds), Wiley, 166-261, 1971.

Holmes et ai, Biopolymers (Peptide Science), 37:199-211, 1995.

Hunter & Sanders, J. Am. Chem. Soc, 112:5525-5534, 1990.

Kaiser et al.,Anal. Biochem., 34:595, 1970.

LaBrenz & Kelly, J. Am. Chem. Soc, 117:1655-1656, 1995.

Laugaa et ai. Biochemistry, 24:5567-5575, 1985.

Lchn, Angew. Chem. Int. Edn. Engl.29:1304-1319, 1990.

Lerman,./. Mol. Biol, 3:18-30, 1961.

Lindsey, New J Chem., 15:153-180, 1991.

Martin etal.,,1. Med. Chem., 38:1431-1436, 1995.

McGhee & von Hippel, J. Mol. Biol., 86:469-489, 1974.

Ohlmeyer et ai, Proc. Natl. Acad. Sci. USA, 90: 10922-10926, 1993.

Pascher et ai , Science, 271 : 1558- 1560, 1996.

Peek et ai, Biochemistry, 33:3794-3800, 1994.

Pci etal., Science, 253:1408-1411, 1991.

Rawls, Chemical and Engineering News, 37-39, 1996.

Revet et ai, Nature New Biol, 229: 10-13, 1971.

Sambrook et ui. Molecular Clonin.g, 2nd ed., Cold Spring Harbor Laboratory Press, CSH, 1.38-1.39, 1989.

Shrederetα/.. J. Am. Chem. Soc, 117:2673-2674, 1995.

Stork et al, Accts. Chem. Res.

Takenaka, et al Supramol Chem., 2: 41-46, 1993.

Wakelin, M<?./. Res. Rev., 6:275-340, 1986.

Waring & Fox, in Molecular Aspects of Anticancer Drug Action, Niedle & Waring (eds), Macmillan, London, 127, 1983.

Waring, Ciba Foundation Sym., 158: 128-142.

Whitesides et al. , Science, 254: 1312-1319, 1991.

Williams et al., Proc Natl Acad. Sci., 87: 2225-2229, 1990.

Wilson & Jones, Intercalation Chemistry, Whittingham & Jacobson (eds), Academic Press, New York, 1982.

Winkler & Gray, Chem. Rev., 92:369-379, 1992.

Wirth et al, J. Am. Chem. Soc, 1 10:932-989, 1988.

Yen et al, Biochemistry, 21 :2070-2076, 1982.