ACKNOWLEDGEMENT This invention was made with U.S. government support under grant number GM 27137 from the National Institute of General Medical Sciences and grant number N00014-92-J-1412(R&T code 413t01 i) from the Office of Naval Research. The U.S. government has certain rights in the invention.

FIELD OF THE INVENTION This invention pertains to chemical modification of polymers. BACKGROUND OF THE INVENTION Functionalized polymers have been the subject of intensive research, owing to their wide potential application in biology, chemistry, medicine, and in techniques involving ion-exchange resins, immobilized biological macromolecules, and electrically conductive polymers. Akelah et al.. Functionalized Polymers and Their Applications . Chapman and Hall, London (1990). Chemical modification of polymer films or film surfaces with concomitant introduction of functional groups is important for the development of new materials such as novel composites, Baum et aL. Chem. Mater. 3:714-720(1991): resist materials, MacDonald et aL. Chem. Mater. 3:435-442(1991): biosensors. Pantano et al.. J. Am. Che . Soc. 113:1832- 1833 (1991); and biomaterials, Allcock et a!.. Chem. Mater. 3:450-454(1991).

Examples of existing methods for modifying polymer films include sulfonation of polystyrene, Gibson et al.. Macromolecules 13:34 (1980): sulfonation of poly(aryloxy)phosphazenes, Allcock et aL. Chem. Mater. 3:1120(1991): plasma treatment of polyester, Porta et aL. Chem. Mater. 3:293 (1991): base hydrolysis of polyimide, Lee et al., Macromolecules 23:2097 (1990): base hydrolysis of polyphosphazenes, Allcock et aL. Chem. Mater. 3: 1441 (1991): and base treatment of poly(vinylidene fluoride), Dias et al., Macromolecules 17:2529 (1984).

Another conventional method for modifying polymers comprises exposing the surface of a hydrocarbon polymer such as polyethylene with nitrene or carbene intermediates generated in the gas phase. Breslow, in Scriven (ed.), Azides and Nitrenes . chapter 10, Academic Press, NY (1984). Also, difluorocarbene generated in solution has been reported to modify 1,4-polybutadienes. Siddiqui et al.. Macromolecules 19:595 (1986).

Perfluorophenyl azides (PFPAs) have been shown to exhibit improved CH- insertion efficiency over their non-fluorinated analogues when the PFPAs were photolyzed in hydrocarbon solvents such as cyclohexar.e or toluene. Keana et al.. J. Fluorine Chem. 43:151 (1989); Keana et aL. J. Org. Chem. 55:3640(1990): Leyva et al.. J. Org. Chem. 54:5938(1989): and Soundararajan et aL. J. Org. Chem. 55:2034 (1990). PFPAs were initially developed as efficient photolabeling reagents. Cai et al.. Bioconiugate Chem. 2:38 (1991): Pinney et al., _, Org. Chem. 56:3125 (1991); and Crocker et al.. Bioconiugate Chem. 1:419 (1990). Recently, bis-(PFPA)s have been shown to be efficient cross-linking agents for polystyrene, Cai et al.,

Chem. Mater. 2:631 (1990): and poly(3-octylthiophene), Cai et aL. J. Molec. Electron. 7:63 (1991).

In view of the present state of the art in chemical modification of polymers, there remains a need for other methods for functionalizing polymers, particularly methods that are easier to perform and more readily adaptable for functionalizing polymers with a wide range of functional groups.

There is also a need for methods for functionalizing polymers that have heretofore been resistant to being chemically modified.

There is also an ongoing need for new types of chemically modified polymers for use in any of a wide variety of specialized applications such as, but not limited to, biocompatible polymer films and other structures, adhesives and adhesive-compatible materials, composite matrices, membranes, insulators for semiconductor materials, fibers, foams, and films.

SUMMARY OF THE INVENTION The foregoing needs are met by the present invention which provides methods for covalently modifying (i.e., functionalizing) various polymeric substances, and provides various functionalized polymers.

Polymeric substances that can be functionalized according to the present invention include any of various substances comprising synthetic and/or natural polymer molecules having chemical moieties each capable of undergoing an addition reaction with a nitrene.

According to the present invention, a polymeric substance is functionalized by adding to the polymeric substance a functionalizing reagent. The functionalizing reagent comprises molecules each having a nitrenogenic group and a functionalizing group. The molecules of the functionalizing reagent are brought into reactive proximity to the polymer molecules such as by, but not limited to, forming a solution of the functionalizing reagent and the polymer molecules. The solution can be formed into a film or other suitable shape, then dried.

While the molecules of the functionalizing reagent and the polymer molecules are in reactive proximity, the molecules are exposed to a reaction-energy source such as photons, electrons, or heat. In the presence of the reaction-energy source, the nitrenogenic groups on molecules of the functionalizing reagent form nitrene intermediates that covalently react with -CH, -NH, -OH, -C=C-, C-C and other groups on the polymer molecules so as to cause "nitrene addition" or "nitrene insertion" of the functionalizing groups to the polymer molecules. The nitrene addition or nitrene insertion results in the functional groups becoming covalently bonded to the polymer molecules.

The nitrenogenic groups on molecules of the functionalizing reagent are azide groups or analogous chemical groups capable of forming a reactive nitrene when exposed

to a reaction-energy source.

According to the present invention, the polymers can be functionalized via either a single-stage or a multi-stage process. In a multi-stage process, each stage typically involves different functionalizing reagents. In both single- and multi-stage processes, at least one stage involves a nitrenogenic functionalizing reagent.

In a single-stage process, each molecule of the functionalizing agent comprises, in addition to the nitrenogenic group, a functionalizing group covalently coupled to the nitrenogenic group. The functionalizing group can be virtually any desired chemical group that does not cross-react with the nitrenogenic group or is geometrically prevented from reacting with the nitrene intermediate. E.g., the functionalizing group can be selected from, but is not necessarily limited to, radioactive labels, fluorescent labels, enzymes, pharmacologically active groups, diagnostically active groups, antibodies, nucleic acids, surfactants, and any of a wide variety of other groups.

Functionalizing reagents adapted to functionalize substrates in multi-stage reactions can be configured in several ways. According to one method, a first functionalizing reagent is reacted with the polymer molecules so as to achieve covalent attachment of the first functionalizing-reagent molecules to the polymer molecules; afterward, a second functionalizing reagent is added so as to react with, and therefore covalently bond to, the attached first functionalizing-reagent molecules. In such a method, the first functionalizing reagent comprises molecules each comprising, in addition to the nitrenogenic group, a first functionalizing group adapted to participate in downstream chemistry after molecules of the first functionalizing reagent have been covalently bonded to the polymer molecules via nitrene addition. For example, the first functionalizing group can be an active ester that is reactive with -NH groups, -OH groups, or other nucleophilic groups on molecules of a second functionalizing reagent. The second functionalizing reagent, then, can provide a second functionalizing group ultimately desired to be attached to the polymer molecules, such as an enzyme, antibody, diagnostic agent, or therapeutic agent.

An alternative multi-stage process comprises first reacting the second functionalizing reagent (comprising the second, or ultimately desired, functionalizing group) with the first functionalizing reagent (including a nitrenogenic group); then, in a second reaction, reacting the product of the first reaction with the polymer molecules in the presence of a reaction-energy source so as to covalently attach the product of the first reaction to the polymer molecules via nitrene addition.

A class of preferred functionalizing reagents for single- and multi-stage processes according to the present invention consists of N.-hydroxysuccinimide active ester- functionalized perfluorophenyl azides (NHS-PFPAs). The NHS active ester groups become covalently attached to the polymer molecules via generation during the reaction of highly reactive nitrene intermediates derived from the PFPA portion of the reagent molecules. (The

reactive nitrene portion of the intermediates are preferably constrained structurally such that the nitrene portion cannot react intramolecularly with the NHS active ester portion.) Thus, the polymer molecules become "modified" (i.e. /functionalized"). Afterward, the active esters can participate in further reactions with a variety of reagents containing primary amines or hydroxyls (such as biomolecules) by way of amide or ester formation, respectively.

According to another aspect of the present invention, a mixture comprising molecules of a nitrene-forming functionalizing reagent and polymer molecules can be applied, such as in the form of a film, to the surface of a substrate. Then, the coating or film is exposed to a reaction-energy source (such as photons or a beam of particles such as an electron beam) in a spatially selective way to functionalize certain regions of the surface and not others, thereby creating a functionalized pattern on the surface. Such patterns can have dimensions measured in micrometers and smaller, due to the highly resolved manner in which the coated surface can -be exposed to the reaction-energy source. Thus, the present invention has wide applicability in microelectronics and in the construction of novel micron-scale biosensors.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows IR spectra of polystyrene including 8 wt-% NHS-PFPA (compound 1 in Scheme 10), wherein plot "a" was obtained before photolysis; plot "b'was obtained after photolysis; plot "c"was obtained after treatment with the amine 3 (Scheme 10); and the peaks at 2300 cm ^"1 are from C0 ₂ .

FIG. 2 shows IR spectra of poly(3-octylthiophene) including 10 wt-% NHS- PFPA (compound 1 in Scheme 10), wherein plot "a" was obtained before photolysis; plot "b" was obtained after photolysis; and plot "c"(shown offset from plot "b")was obtained after treatment with the amine 3 (Scheme 10). FIG. 3A is a photomicrograph obtained using an optical microscope, depicting linear and circular functionalized patterns produced on a film of polystyrene and 8 wt-% NHS-PFPA by electron-beam lithography.

FIG. 3B is a photomicrograph obtained using a fluorescence microscope fitted with a fluorescein filter set, depicting the functionalized patterns of FIG. 3A after treatment with amino-fluorescein.

FIG. 4 A is a photomicrograph obtained using a fluorescence microscope of circular patterns produced on a film of poly(3-octylthiophene) and 7 wt-% of NHS-PFPA by exposing the film to electron-beam lithography conditions and subsequently treating the film with amino-fluorescein, wherein the microscope was fitted with a rhodamine filter set. FIG. 4B is a photomicrograph obtained using a fluorescence microscope of circular patterns produced on a film of poly(3-octylthiophene) and 7 wt-% of NHS-PFPA by exposing the film to electron-beam lithography conditions and subsequently treating the film with amino-fluorescein, wherein the microscope was fitted with a fluorescein filter set.

FIG. 4C is a photomicrograph obtained using a fluorescence microscope of circular patterns produced on a film of ρoly(3-octylthiophene) by exposing the film to electron- beam lithography conditions and subsequently treating the film with amino-flucrescein, wherein the microscope was fitted with a rhodamine filter set. FIG. 4D is a photomicrograph obtained using a fluorescence microscope of circular patterns produced on a film of poly(3-octylthiophene) by exposing the film to electron- beam lithography conditions and subsequently treating the film with amino-fluorescein, wherein the microscope was fitted with a fluorescein filter set.

DETAILED DESCRIPTION The following terms are used herein:

A "polymeric material" is a material comprising polymer molecules or a network of polymer molecules.

A "polymer molecule" is a relatively large molecule formed by the covalent linking together of smaller molecules termed "monomers/ The monomers present in a polymer molecule can be the same or different. Polymer molecules can be natural, such as (but not limited to) any of various polysaccharides and polypeptides; or synthetic such as (but not limited to) nylon and polyethylene. In a polymeric material, polymer molecules can be associated with each other in any of several ways, including non-covalently (as a thermoplastic) or a covalently cross-linked network (as a thermoset). A "functionalized polymer" can pertain to either a functionalized polymeric material or a molecule of a functionalized polymeric material. Functionalized polymer molecules comprise one or more functional groups covalently bonded thereto according to the present invention.

A "functional group" is a group of one or more atoms bonded together in an organized way so as to have a desired chemical property. Certain functional groups can, when covalently bonded to a polymer molecule according to the present invention, participate in one or more additional bonding reactions with either a similar functional group or a different type of functional group. Such bonding reactions can result in: (a) attachment to the functional groups of any of a variety of additional functional groups; or (b) coupling together (cross- linking) of the functionalized polymer molecules. Many other functional groups attachable to polymer molecules according to the present invention can confer altered chemical properties to the polymer molecules such as, but not limited to, making them labeled or tagged with a fluorescent, radioactive, immunologic, diagnostic, or therapeutic marker.

A "functionalizing reagent" according to the present invention is a reagent adapted for functionalizing a polymer according to the present invention. Molecules of functionalizing agents have at least one nitrenogenic group (as a first functional group) coupled to a second functional group, wherein the nitrenogenic group is preferably constrained by the functionalizing-reagent molecular structure between the nitrenogenic group and the functional

group The nitrenogenic groups are capable under reaction conditions of functionalizing polymer molecules.

A "nitrenogenic group" on a functionalizing reagent is a chemical moiety that, when exposed to a reaction-energy source, becomes a nitrene group. A "nitrene group" (also generally termed "nitrene" or "nitrene intermediate") is a particular form of nitrogen group that can be depicted as a singlet by the structure: R-& and as a triplet by the structure: R-N'. Nitrenes are regarded by persons skilled in the art as the nitrogen analogs of carbenes. Like carbenes, nitrenes are generally regarded as intermediates. Nitrenes are highly reactive and generally cannot be isolated under ordinary conditions. However, certain chemical reactions such as reactions according to the present invention would not otherwise be explainable by known reaction mechanisms without the presumed existence of nitrenes. Important nitrene reactions can be summarized by the following:

(a) Nitrenes, including aryl nitrenes, can undergo addition reactions at -CH sites and at -NH sites; e.g.:

Ar-N. + R ₃ C-H -* Ar-NHCR ₃

Ar-N R ₂ N-H →Ar-NHNR ₂

(b) Nitrenes can also undergo addition at -C-C- and -C=C- bonds; e.g.

R-N ^~ + R,C=CR, →R,C CR,

^' \ /

N

R

As used herein, the term "addition reaction" when used in the context of reactions of the nitrene group of the functionalizing reagent with polymer molecules, generally refers to any of the various addition and insertion reactions that nitrenes can undergo with polymer molecules according to the present invention.

According to the present invention, a functionalizing reaction occurs when a functionalizing reagent comprising a nitrenogenic group is exposed to a reaction-energy source, which converts the nitrenogenic group to a nitrene intermediate. The functionalizing reaction proceeds by reaction of the nitrene intermediate with a polymer molecule.

A "reaction-energy source" is an energy source that drives a functionalizing reaction according to the present invention by, in particular, converting nitrenogenic groups on

functionalizing reagent molecules to nitrenes which react with the polymer molecule. Suitable reaction-energy sources include (but are not limited to): photons (such as ultraviolet (UV) light, deep-UV light, laser light, X-rays, and heat in the form of infrared radiation or conductive heating), energized electrons (such as an electron beam), and energized ions (such as an ion beam). These reaction-energy sources are conventionally used for such tasks as lithography, scanning microscopy, and, in the case of UV and visible photons, effecting photochemical reactions and excitation of fluorescent molecules.

A "functionalizing reaction" is a reaction in which polymer molecules are functionalized according to the present invention. A functionalizing reaction can consist of one or more stages. At least one stage involves the reaction in the presence of a reaction-energy source of the polymer molecules with molecules of a functionalizing reagent comprising nitrenogenic groups.

According to the present invention, a polymer molecule is functionalized by a chemistry whereby functional groups on functionalizing reagent molecules become covalently bonded to the polymer molecule. Such covalent bonding is achieved by conversion of nitrenogenic groups on the functionalizing reagent molecules (the functionalizing reagent molecules also each comprising a desired functional group as set forth below) to a nitrene intermediate highly reactive with the polymer molecule by exposure of the functionalizing reagent molecules to a reaction-energy source. The functionalizing reagent is preferably selected from a group consisting generally of: aryl azides, alkyl azides, alkenyl azides, alkynyl azides, acyl azides, and azidoacetyi derivatives, all capable of carrying a variety of substituents. Most preferably, fluorine (and/or chlorine) atoms are present to the maximum extent possible in the positions on the functionalizing reagent molecule adjacent the azide group. Each of the foregoing azides may also contain within the same molecule any of the following functional groups, constrained structurally from reacting with the nitrene moiety after the nitrene moiety is generated:

(a) carboxyl groups and various derivatives thereof such as (but not necessarily limited to): N.-hydroxysuccinimide esters; N.-hydroxybenztriazole esters; acid halides corresponding to the carboxyl group; acyl imidazoles; thioesters; p_-nitrophenyl esters; alkyl, alkenyl, alkynyl and aromatic esters, including esters of biologically active (and optically active) alcohols such as cholesterol and glucose; various amide derivatives such as amides derived from ammonia, primary, and secondary amines and including biologically active (and optically active) amines such as epinephrine, dopa, enzymes, antibodies, and fluorescent molecules; (b) alcohol groups, either free or esterified to a suitable carboxylic acid which could be, for example, a fatty acid, a steroid acid, or a drug such as naprosin or aspirin;

(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as a carboxylate anion, thiol anion, carbanion, or alkoxide ion, thereby

resulting in the covalent attachment of a new group at the site of the halogen atom;

(d) maleimido groups or other dienophilic groups such that the group may serve as a dienophile in a Diels-Alder cycloaddition reaction with a 1 ,3-diene-containing molecule such as, for example, an ergosterol; (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of well-known carbonyl derivatives such as hydrazones, semicarbazones, or oximes, or via such mechanisms as Grignard addition or alkyllithium addition; and

(f) sulfonyl halide groups for subsequent reactions with amines, for example, to form sulfonamides. The foregoing functional groups are particularly adapted for participation in downstream chemistry (i.e., chemistry performed after the foregoing functional groups are attached to the polymer molecules) whereby yet other functional groups can be covalently attached to the polymer molecules by reaction with the already-attached functional groups.

A general reaction by which a representative functionalizing reagent is converted to a nitrene intermediate is:

X-R-N ₃ X-R-N + N, photons or e ^" beam

where X is the functional group, N ₃ is the nitrenogenic group (an azide in this instance), and R is an aromatic ring, heteroaromatic ring, or other carbon-containing fragment.

A reaction-energy source comprising UV light can be supplied to the reaction by, for example, one of the following representative procedures: (a) A sample comprising functionalizing reagent molecules and polymer molecules is placed in a well of a Rayonet Photochemical Reactor fitted with either 350-nm, 300-nm, or 254-nm lamps and irradiated at ambient temperature for several minutes under air. The duration of the irradiation can be adjusted to change the exposure dose, (b) The sample is irradiated through a high-resolution photomask, for example, by (but not limited to) projection UV lithography. (c) Photolysis is carried out in a KSM Karl Suss deep-UV contact aligner using a contact high-resolution photomask. It will be readily appreciated by persons skilled in the art that such procedures can also be generally used to provide the functionalizing reaction with photons of wavelengths other than UV.

A reaction-energy source comprising electrons can be supplied to the reaction by the following representative procedure: A film sample comprising functionalizing reagent molecules and polymer molecules is irradiated under vacuum by an electron or particle beam with an energy selected within the range 1-40 kV. (A representative electron-beam source is a JEOL 840A electron microscope modified for electron-beam lithography.) The beam is stepped across the film surface to expose certain areas and not others. A dwell time

at each step can be adjusted to change the exposure dose.

Particularly effective functionalizing reagents are selected from the group of perfluorophenyl azides (PFPAs) derived from 4-azido-2,3,5,6-tetrafluorobenzoic acid in which the carbonyl group is further activated through reactive ester, amide, acid halide, or mixed anhydride formation.

For example, and not intended to be limiting, representative functionalized perfluorophenyl azides have the general structure:

wherein X can be any of the following: CN, CONH ₂ , CHO, C0 ₂ Me, COMe, N0 ₂ , C0 ₂ H, COC1, CO-Imidazole, CONHS, CH ₂ OH, CH ₂ NH ₂ , COCH ₂ Br, N-maleimido, NH-biotinyl, CONH-R (where R is a polypeptide moiety), CONH-X-S-S-Y-NH-biotinyl (where X and Y are spacer atoms and the S-S bond is reductively cleavable at a later stage), and CONHS-S0 ₃ Na.

Representative activated PFPAs include (but are not limited to) the N_- hydroxysuccinimide (NHS) ester A (also designated "NHS-PFPA"), the p_-nitrophenyl ester B, the 1-hydroxybenzotriazole ester C, the acyl imidazole D, the acid chloride E, the mixed anhydride F and the 2,2,2-trichloroethyl ester G:

B

In addition to the foregoing candidate functionalizing reagents, it is possible to utilize other PFPAs having "spacers" situated between the reactive functional group and the PFPA moiety, such as:

Other candidate aryl azides useful as functionalizing reagents are similar to the above examples except that another aryl moiety replaces the PFPAj such as:

Candidate polymers that can be functionalized according to the present invention include virtually any polymer comprising polymer molecules possessing -CH groups, -

NH groups, -OH groups, C-C sites, and/or -C=C- sites. Such polymers include, but are not limited to: (a) saturated polyolefins as exemplified by polyethylene, polyvinyl chloride, polytetrafluoroethylene, polypropylene, polybutenes, and copolymers thereof;

(b) acrylic resins such as polymers and copolymers of acrylic acid, methacrylic acid [poly(methylmethacrylate), poly(hexylmethacrylate)], and acrylonitrile;

(c) polystyrene and its analogues such as poly(ρ_-chlorostyrene) and poly(p_- hydroxystyrene);

(d) unsaturated polyolefins such as poly(isoprene) and poly(butadiene);

(e) polyimides such as polyimide(benzophenone tetracarboxylic dianhydride/tetraethylmethylenedianiline);

(f) polyesters such as poly(trimethylene adipate) and poly(hexymethylene sebacate);

(g) conjugated and conducting polymers such as poly(3-alkylthiophene), poly(3-alkylpyrrole), and polyaniline;

(h) inorganic polymers such as poly(aryloxyphosphazene), polvfbis(trifluoroethoxy)phosphazenel, polysilanes, and polycarbosilanes, siloxane polymers, and other silicon-containing polymers;

(i) organic metals (i.e., organic polymers with metallic properties) such as polycroconaines and polysquaraines, as described in Chemical and Engineering News (August 31, 1992), p.8.

(j) organometa'lic polymers such as palladium poly-yne and ferrocene- containing polyamides; and

(k) polysaccharides such as cellulose fibers, chitin, and starch. Functionalization of polymer molecules according to the present invention requires that molecules of the functionalizing reagent and the polymer molecules be brought into "reactive proximity";i.e., brought together sufficiently closely so as to undergo a functionalizing reaction when exposed to the reaction-energy source. One way in which this can be done is to prepare a solution comprising the polymer molecules and the functionalizing reagent. Another way is to prepare a suspension or mixture comprising the functionalizing reagent and polymer particles or agglomerations of the polymer. Yet another way is to apply the functionalizing reagent (such as a solution of the functionalizing reagent in a solvent capable of absorbing into the polymeric material) to a surface of the polymer, then allow the functionalizing reagent to absorb into the polymeric material.

Functionalization of a polymer can occur in one or more stages, depending upon various factors such as the particular polymer to be functionalized; the form of the polymer (i.e., solution, particulate suspension, non-fluid mass); the functional group(s) to be attached to the polymer molecules; the necessity to protect the functional groups from undesired reactions during reaction of the functionalizing reagent with the polymer molecules; and on other matters. For example, in a one-stage functionalization, polymer molecules and molecules of a functionalizing reagent each having a nitrenogenic group and a desired functional group are brought into reactive proximity. Upon exposure to a reaction-energy source, the nitrenogenic groups are converted to nitrenes which react with -CH, -NH, -OH, -C=C-, C-C, and other groups on the polymer molecules reactive with nitrenes, thereby covalently bonding the functional groups to the polymer molecules. The functional groups typically do not require additional chemistry performed on them to confer the desired useful property to the resulting functionalized polymers.

In a two-stage functionalization protocol, each stage involves a different functionalizing reagent. In many instances, the first stage can be performed by interspersing molecules of a first functionalizing reagent depthwise into the polymer mass, such as by first forming a fluid solution or suspension comprising the polymer and the first functionalizing reagent; forming the fluid into a desired shape; then converting the fluid into a product having a rigid form. The reaction-energy source is then applied to the rigid product to covalently bond the first functionalizing reagent to the polymer molecules. Subsequently, during the second stage, the second functionalizing reagent is applied to a surface of the rigid product.

As an example of a two-stage functionalization reaction, the first stage involves a first functionalizing reagent such as an NHS-PFPA compound. Upon exposure to a reaction-energy source, the azide group of the PFPA portion is converted to a nitrene

intermediate that reacts with polymer molecules. Thus, the NHS active-ester groups on the NHS-PFPA molecules become covalently attached to the polymer molecules by a reaction that can be generally indicated as shown in Scheme 1 (wherein a polymer molecule is represented by a circumscribed P):

1 * Scheme 1

As can be seen, the NHS-ester portions of the PFPAs do not participate in this first-stage chemistry. Rather, the NHS-esters, after being transferred to the polymer molecules, are utilized in second-stage chemistry, discussed below.

In the second stage, the NHS-esters readily react with molecules of a second functionalizing reagent. The second functionalizing reagent is selected from a group consisting of molecules possessing primary or secondary amines and/or hydroxyls. Reaction of NHS-esters with primary amines proceeds via amide formation as shown in Scheme 2.

Scheme 2 wherein compound 2 is as shown in Scheme 1. Reaction of NHS-esters with hydroxyls proceeds via ester formation, as shown in Scheme 3.

Scheme 3 wherein compound 2 is as shown in Scheme 1.

Since many types of biological molecules possess amine and/or hydroxyl groups, these molecules can serve as functionalizing reagents adapted for reaction in a second- stage functionalization reaction with NHS-esters covalently bonded to the polymer molecules in a first-stage functionalization reaction. Thus, it is possible to attach any of a wide variety of molecules, including macromolecules such as proteins, nucleic acids, carbohydrates, and various other molecules, to polymers using methods according to the present invention.

It is also possible according to the present invention to first prepare nitrenogenic derivatives of molecules (such as biomolecules, drugs, analytes, catalysts [including transition metals], and diagnostic agents) to be attached to the polymers, bring the derivatives into reactive proximity with the polymer molecules, then expose to a reaction-energy source to cause the nitrenogenic derivatives to covalently bond to the polymer molecules via nitrene intermediates. It is necessary that the nitrenogenic moiety be structurally constrained to prevent the nitrene from readily reacting with another part of the same molecule. Thus, with NHS-PFPA functionalizing reagents, the 4-position of the phenyl ring is the preferred position for the azide group. To convey the scope of the present invention without intending in any way to be limiting, the following representative functionalizations according to the present invention are provided:

(a) Carcinogenic or mutagenic polycyclic aromatic hydrocarbons c-in be attached to polymer molecules to render the polymers "carcinogenic." Candidate polycyclic hydrocarbons include ethidium compounds and various pyrene compounds (such as 1- pyrenemethylamine and 6-aminochrysene). It is also possible, when attaching such compounds to polymer molecules, to employ "spacer groups" serving to "lift"the hydrocarbon from the polymer molecule. A representative spacer-containing hydrocarbon is the primary amine derived from 1-pyrenebutyric acid. Such reactions can be depicted generally as shown in Scheme 4.

Scheme 4 wherein 2 is as shown in Scheme 1 and Z represents a spacer group.

(b) The hydrophobicity of a polymeric material can be altered, after attachment of NHS-ester groups to the polymer molecules in a first-stage reaction (via a nitrene intermediate), by subsequent reaction of the NHS-ester groups with long-chain aliphatic amines such as 1-aminohexadecane in a second-stage reaction. Such a reaction can be generally depicted as shown in Scheme 5:

Scheme 5 wherein R is a chain of hydrophobic atoms such as, for example, C 12^25-. oleyl, octadecyl, 3-jS-aminocholestane, or hexyldimethylsilyl;and 2 is as shown in Scheme 1. (c) The hydrophilicity of the polymer can be altered, after attachment of

NHS-ester groups to the polymer molecules in a first-stage reaction (via a nitrene intermediate), by subsequent reaction of the NHS-ester groups with amine-possessing highly polar molecules in a second-stage reaction. Such amine-possessing polar molecules include (but are not necessarily limited to): glucosamine, ethanolamine, polyethyleneimine (protonated at pH 7), polylysine (also protonated at pH 7), glycerol, and other polyhydroxy compounds.

Such reactions can be generally depicted as shown in Scheme 5 but wherein R is HOCH ₂ CH ₂ -, or NH _j ^^CH _j NH-^-CH _j CH _j -; and 2a and 2b are as shown in Scheme 1. For polyalcohols, such reactions can be generally depicted as shown in Scheme 6:

Scheme 6 wherein R is, for example, CH-CHOH-CH ,OH; and 2 is as shown in Scheme 1.

(d) The polymer can be made surface-active by first attaching NHS-ester groups to polymer molecules in a first-stage reaction. The reaction to make the polymer molecules surface-active proceeds by a second-stage reaction employing any of various animated or hydroxylated "detergent" molecules such as, for example, 1-amino-dodecanoic acid. At pH 7 and after attachment of this compound to a polymer molecule, the carboxyl group is ionized and the compound extends away from the polymer molecule as a long hydrophobic tail terminating in a polar carboxylate anion. Such reactions can be generally depicted as shown in Scheme 7.

Scheme 7

wherein R is -(CH _j ^-CO _j H; and 2 is as shown in Scheme 1.

(e) Enzymes and other polypeptides can be attached to polymer molecules previously functionalized in a first-stage reaction with, for example, an NHS active ester. The subsequent second-stage reaction proceeds by, for example, a reaction of a lysine amino group present on the polypeptide molecules with the NHS active ester. A representative reaction is depicted as shown in Scheme 8:

F F

Scheme 8 wherein the circumscribed E with attached NH ₂ group represents a polypeptide comprising a lysine residue. Examples of such a polypeptide include (but not limited to) an enzyme (e.g., horseradish peroxidase), lectin, or antibody. Compound 2 is as shown in Scheme 1.

(f) Antibodies, lectins, and other proteins can also be attached to polymer molecules by functionalizing reactions similar to such reactions for attaching enzymes. Such attached molecules can then be used, for example, as highly selective sensing agents in biosensors.

(g) Specialized molecules can be attached to polymer molecules to control the wettability of the polymer or alter the ability of living cells to adhere to the polymer.

(h) Polymer molecules can be biotinylated in a one or two-stage reaction, followed by treatment of the biotinylated molecules with, for example, a derivatized avidin or streptavidin. The avidin or streptavidin are thus used as bridging units for subsequent attachment of other biomolecules to the polymer. Representative reactions are as follows:

Two-stage reaction (Scheme 9)

Scheme 9

wherein 2 is as shown in Scheme 1 and RNH -_ represents the amino group of N- biotinylhexylenediamine:

A one-stage reaction is exemplified by bringing the polymer molecules and molecules of the PFPA derivatives of biotin:

into reactive proximity, followed by exposure to photolysis or an electron beam.

To further illustrate and describe the present invention, the following examples are provided: Example 1

In this Example, we functionalized the hydrocarbon polymer polystyrene (PS) by -CH insertion of photochemically generated nitrene intermediates.

Referring to Scheme 10, the active-ester azide 1 was formed by esterification of N-hydroxysuccinimide (NHS) with 4-azido-2,3,5,6-tetrafluorobenzoic acid. The active-ester azide 1 was selected for study as a representative functionalizing agent because NHS esters react readily with amine-containing reagents to form amides (Rl-NH-COR).

1

2 + 5-(aminoac ^β tamido)flυorescein

Scheme 10

A solution containing 50.2 mg PS (mean molecular weight 125,000to 250,000daltons) and 4.0 mg NHS ester 1 in 1.0 mL xylene was prepared, yielding an 8 % w/w solution of 1. The solution was spin-coated on a NaCl disc using a photoresist spinner (Headway Research, Inc Gariand, Texas) set at 1000 rpm. After drying the disc at 50 °C for one hour, the film remaining on the disc had a thickness of about 0.7 μm, as measured using

an ellipsometer (Rudolph Research, Inc. , Flanders, New Jersey). The film was photolyzed for 1.5 minutes using a Rayonet photoreactor (Southern New England Ultraviolet Co., Branford, Connecticut) employing 254-nm lamps as photon sources.

The photolysis resulted in the smooth decomposition of the azide group with concomitant formation of the functionalized PS 2 derived from a CH-insertion reaction. The photolysis was monitored by the disappearance of the azide absorption at 2124 cm ^"1 , as indicated in FIG. 1 by comparing curves "a" (before photolysis) and "b" (after photolysis). The active ester carbonyl absorption around 1750 cm ^"1 was not affected by the photolysis reaction. Next, the functionalized PS film 2 was further functionalized by immersion at room temperature for over two hours in a solution of 5.4 mg 4-azido-2,3,5,6- tetrafluorobenzylamine (3) (i.e., the hydrochloride salt of 3) and 10 mg Et ₃ N in nitromethane. (Nitromethane is a solvent that does not dissolve PS.) The film was then removed from the solution and immersed in 40 mL nitromethane for 10 minutes, rinsed using nitromethane, then air dried. The coupling reaction that occurred during said immersion of the functionalized PS film 2 in the solution of 3 was monitored by IR spectroscopy using a Nicolet Model 5DXB FTIR spectrometer (Madison, Wisconsin).

As the coupling reaction proceeded, an azide-absorption peak at 2121 cm ^"1 reappeared because, as the amine 3 attached to the functionalized PS 2, the azide group of 3 remained attached and unreacted. A corresponding decrease in absorption at 1750 cm" ¹ was attributed to loss of the carbonyl groups (>C=0) of the active ester, as also seen in FIG. 1 by comparing curve "c'with curve "b". The IR spectra confirmed that amine 3 reacted with the NHS active esters of the film 2, resulting in the further modification of the PS by incorporation of the perfluorophenylazide groups along the PS polymer chain to yield a functionalized PS polymer 4. IR intensity comparison of the azide absorptions (comparison of curve "c" with curve "a" of FIG. 1) indicated that about 40 percent of the original number of azide groups became incorporated into the PS chain of polymer 4 as a result of treatment of 2 with 3. This was probably due to the fact that photolysis of azide 1 in the presence of PS resulted in less than a 100-percent yield of CH insertion. It is also possible that some of the NHS groups may have been cleaved by adventitious hydrolysis during the treatment with the solution of amine 3 in nitromethane.

Examples 2 and 3

These Examples comprise control experiments for Example 1. Compounds are as shown in Scheme 10.

In Example 2, a solution of PS was prepared as in Example 1 but without NHS active ester 1. The PS solution was formed into a film and photolyzed as in Example 1, then treated with a solution of the amine 3 in nitromethane. Afterward, no azide absorption was observed in the IR spectrum of the film.

In Example 3, a film of PS containing active ester 1 was prepared as in Example 1. The Example-3 film was not photolyzed but rather treated directly with a solution of the amine 3 in nitromethane. IR spectrophotometry revealed the disappearance of absorption at 2124 and 1750 cm ^"1 , showing that the nitromethane had extracted essentially all of the active ester 1 or the corresponding amide out of the polymer.

Examples 2 and 3 showed that both the NHS active ester 1 and photolysis are needed for the modification of the PS film with NHS active ester groups. Example 4

In this Example, referring further to Scheme 10, N-succinimidyl 4-amino- tetrafluorobenzoate (5) was used as a model for the polymer 2. To prepare 5, a mixture of 214 mg (l.OO mmol) 4-amino-tetrafluorobenzoic acid, 119 mg (l.OO mmol) N-hydroxysuccinimide and 211 mg (l.OO mmol) dicyclohexylcarbodiimide in 10 mL CH ₂ C1 ₂ was stirred for 24 hours. The mixture was filtered and the solid was dried. The solid was then stirred with 6 mL acetone and the mixture was filtered. The filtrate was evaporated to leave 262 mg (83 percent yield) of 5 as a white solid having a melting point of 200-201 °C. *H NMR: δ2.899(s, 4), 4.665(s, 2). IR: 3522, 3418, 1779, 1749, 1683, 1530, 1507, 1317 cm" ¹ . MS: 306 (M ⁺ , 2), 192 (100), 164 (30). A mixture of 11 mg (0.036 mmol) of the active ester 5 and 6.9 mg (0.031 mmol) of amine 3 in CDC1 ₃ was prepared and allowed to react (not all the yield of 5, prepared above, dissolved in CDC1 ₃ ). Progress of the reaction was monitored by Η NMR spectroscopy at room temperature. As the reaction progressed, new signals at 54.7 (d) were observed. After 24 hours, a clear solution was obtained. When the reaction mixture was assayed by ^! H NMR spectrometry, no greater amount of signal was seen at 83.941 for 3 and 52.899 for 5.

The mixture was separated by preparative thin-layer chromatography (hexane-THF 1: 1) to give 12 mg (94 percent yield) of the amide 6 as a white solid having a melting point of 155-156 °C (actually a decomposition temperature). Η NMR: M.286(s, 2), 4.701 (d, 2), 6.402 (m, 1). IR: 3411, 2122, 1686, 1668, 1497, 1314, 1239 cm ^"1 . MS: 411 (M ⁺ , 1), 383 (20), 192 (100), 164 (18). The IR spectrum of the amide 6 showed an azide absorption peak at 2124 cm ^"1 , which was also observed in the polymer film of Example 1 after photolysis and reaction with amine 3.

Example 5

In this Example, shown generally in Scheme 11, we investigated the functionalization of the conductive polymer poly(3-octylthiophene) (abbreviated P30T). P30T can be photochemically cross-linked by bJ£-PFPA and can be used for the direct production of conductive structures via cross-linking under electron-beam lithographic conditions. Cai et al., J. Mo . Electron. 7:63 (1991).

Scheme 11

For this Example, the P30T was prepared from 3-octylthiophene as reported in Cai et al.,]d_.

Referring to Scheme 11, a solution of 25.8 mg P30T and 2.6 mg (10 % w/w) of the NHS ester 1 in 0.8 mL xylene was spin-coated on a NaCI disc, dried, photolyzed, and developed as described in Example 1. The photolysis reaction yielded a functionalized polymeric film 9. The film 9 was treated with the amino azide 3 (structure shown in Scheme 10) in nitromethane under conditions as described in Example 1 for treating PS. A

fiinctionalized polymeric film 10 formed which involved an amide-formation reaction between 3 and the NHS active esters with concomitant covalent attachment of a new set of azide groups to the P30T polymer. (In FIG. 2, compare curve "b"with curve "c".) The IR spectrum of the film 10 showed a moderately strong absorption at 2121 cm ^"1 for the azide group (FIG. 2). It is believed that the C-H insertion reaction yielding the functionalized polymer 9 occurred along the octyl side chains without involvement of the thiophene ring. This is based upon the observation that photolysis of the simple PFPA ester methyl 4-azido- tetrafluorobenzoate in cyclohexane/thiophene yielded methyl (N-cyclohexyl-4-amino)- tetrafluorobenzoate as the only CH-insertion product that could be isolated. Example 6

This Example is a control experiment for Example 5.

A solution of 23.2 mg P30T in 0.8 mL xylene (in the absence of 1) was treated with the amine 3 as described in Example 5. No azide absorption was observed in the IR spectrum of the resulting film, indicating that no incorporation of the amine 3 occurred. Therefore, a first functionalization of P30T with a compound such as 1 is necessary in order to perform a second functionalization with the amine 3. Example 7

In this Example, we investigate the use of electron-beam lithography to accomplish both cross-linking of a polymer (i.e., PS) and the introduction of NHS active ester groups in the polymer in a single step. General reactions are illustrated in Scheme 10.

A solution of 50.2 mg of PS and 4.0 mg of NHS ester 1 (8 % w/w) in 1.0 mL xylene was spin-coated on a silicon wafer as described generally in Example 1. The film was dried for 35 minutes at 90 °C and exposed to an electron beam using a scanning electron microscope (SEM) (manufactured by JOEL-SEM, Peabody, Maryland), modified for electron- beam lithography. Nabity et al.. Rev. Sci. Instrum. 60:27 (1989). The electron beam was used to "draw" micron-sized patterns (in the form of eight five-line patterns and a pattern of five circles of different diameters) on the film. The exposed film was "developed" by dipping in xylene for 35 seconds, rinsing in isopropyl alcohol for 10 seconds, then drying with a stream of nitrogen, thereby yielding a "developed" film 2. The film 2 was photographed using an optical microscope, yielding results shown in FIG. 3A.

In FIG. 3A, the widths of the lines in each five-line set were 0.1,0.2,0.5, 1.0, and 2.0 μm. Each successive five-line set was obtained with an increased electron-beam intensity relative to the preceding set. In the top row of sets, the electron-beam intensities were 50, 60, 70, and 80 pC/cm ~. In the bottom row of sets, the electron-beam intensities were 90, 100, 110, and 120 μC/cm ^~ ~. The line width of each of the circles was the same: 0.5 μm. The electron-beam intensity used to "draw" the circles was 60 μC/cm ² .

The lines and circles shown in FIG. 3 A are composed of functionalized polystyrene 2 (i.e., polystyrene molecules having active esters covalently bonded thereto).

-99-

Referring now to Scheme 10, the film 2 (after obtaining the photographs shown in FIG. 3A) was immersed in a solution of 2.5 mg of amino-fluorescein (compound 7) and 8.3 mg of Et ₃ N in 1.5 mL of EtOH for 4 hours so as to introduce an easily visible fluorescent marker at the active-ester sites on the film. Afterward, the film was washed with EtOH, immersed in EtOH for 2 hours, rinsed with EtOH, then air-dried to yield the film 8. The film 8 was observed under a fluorescence microscope (Carl Zeiss, Germany) equipped with epifluorescence optics. The microscope was fitted with a fluorescein filter set (excitation wavelength 450-490 nm, emission wavelength 515-565 nm). The fluorescence patterns shown in FIG. 3B were observed. Since the fluorescent patterns exhibited by film 8 (FIG. 3B) were coincidental to the patterns observed of the functionalized polystyrene 2 shown in FIG. 3A, we concluded that coupling of the fluorescence marker in film 8 occurred only at sites (film 2) on the polymer to which active esters had been previously coupled.

In FIG. 3B, functionalization of PS had occurred at a dosage of about 50 μC/cm ~- in the film. We also found that crosslinking of PS alone required about 90 μC/cm ² . Example 8

This Example is an experimental control for Example 7. Compounds are as shown in Scheme 10.

A PS film was prepared without the NHS ester 1 but otherwise treated as described in Example 7. The film was exposed to an electron beam and developed as described in Example 7 and photographed using an optical microscope. The PS film was then treated with amino-fluorescein 7 and observed under a fluorescence microscope. No fluorescence pattern was observed. Therefore, prior attachment of the NHS active ester 1 to the PS molecules was required for the subsequent attachment of the amino-fluorescein label 7 to the polymer. Example 9

This Example is similar to Example 3 except that, in this Example, we "drew" micron-sized patterns on a P30T film containing NHS active ester using an electron beam. The general reactions are shown in Scheme 11. A solution of 25.7 mg of P30T and 1.8 mg of NHS ester 1 (7 % w/w) in 0.6 mL of xylene was spin-coated on a silicon disc and dried at 60°C for 30 minutes. The resulting film was exposed to an electron beam as described in Example 3 so as to "draw" micron-sized patterns on the film (line width 0.5 μ ; beam intensity 20 μC/cm ~). The film was then "developed" by dipping in xylene for 10 seconds, rinsing in isopropyl alcohol for 10 seconds and drying under a stream of nitrogen gas to yield the film 9. The film was then immersed in a solution of 1.5 mg of amino-fluorescein 7 and 6 mg of Et ₃ N in 1 mL of EtOH for 4 hours. The film was then washed with EtOH, immersed in EtOH for 1 hour, washed again with EtOH, then air-dried to produce the sample film 11.

The sample film 11 was observed and photographed using a fluorescence microscope equipped with a rhodamine filter set (excitation wavelength 510-560 nm, emission wavelength > 590 nm), yielding the results shown in FIG. 4A. The same sample film was observed and photographed using the fluorescence microscope equipped with a fluorescein filter set (excitation wavelength 450-490 nm, emission wavelength 515-565 nm) yielding the results shown in FIG. 4B. As can be seen, substantially identical patterns were observed having strong fluorescence at both the rhodamine excitation wavelength (FIG. 4A) and the fluorescein excitation wavelength (FIG. 4B).

P30T alone is strongly fluorescent at the rhodamine excitation wavelength but only weakly fluorescent at the fluorescein excitation wavelength. (This is why the films in this Example were observed using a rhodamine filter set and a fluorescein filter set; strong fluorescence observed at the fluorescein excitation wavelength would necessarily be due to the presence of other molecules than just P30T.) In FIGS. 4A and 4B, the observed strong fluorescence at both the rhodamine and fluorescein excitation wavelengths indicates that fluorescein became attached to the regions exposed to the electron beam (FIGS. 4A and 4B). Example 10

This Example is a control for Example 9.

A P30T film (without the active ester 1) was exposed to an electron beam (intensity 30 μC/cm ² , line width 0.5 μm). developed, then treated with amino-fluorescein 7 as described in Example 9. The micron-sized patterns "drawn" on the control P30T film were identical to the patterns in Example 9. When the control film was examined using a fluorescence microscope, strong fluorescence was observed at the rhodamine excitation wavelength (FIG. 4C), but only weak fluorescence was observed at the fluorescein excitation wavelength (FIG. 4D). The results indicate that substantially no fluorescein 7 became attached to

P30T in the absence of activated ester groups. Therefore, the presence of NHS active ester is required in order to obtain any substantial covalent coupling of the fluorescein 7 to P30T. Example 11

In this Example, we functionalized poly(3-octylthiophene) (P30T) as shown in Scheme 12.

Scheme 12 In this scheme, a solution of NHS-PFPA (2) and P30T (1) was spin-coated onto the surface of a silicone substrate in a manner as generally discussed above, then exposed to a reaction-energy source such as 254-nm photons or an electron beam to yield the functionalized P30T (3). Subsequent reaction of the functionalized P30T 3 with the PFPA compound 4 produced 5. Reaction of 5 with aminoacetamidofluorescein yielded fluoroescein- labeled P30T (6).

While the invention has been described in connection with preferred embodiments and multiple examples, it will be understood that it is not limited to those embodiments. On the contrary, it is intended to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.