EP0758250A1 - Supports solides de la texaphyrine et dispositifs associes - Google Patents

Supports solides de la texaphyrine et dispositifs associes

Info

Publication number
EP0758250A1
EP0758250A1 EP95920377A EP95920377A EP0758250A1 EP 0758250 A1 EP0758250 A1 EP 0758250A1 EP 95920377 A EP95920377 A EP 95920377A EP 95920377 A EP95920377 A EP 95920377A EP 0758250 A1 EP0758250 A1 EP 0758250A1
Authority
EP
European Patent Office
Prior art keywords
iii
texaphyrin
matrix
supported
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95920377A
Other languages
German (de)
English (en)
Inventor
Jonathan L. Sessler
Brent L. Iverson
Vladimir Kral
Richard E. Thomas
Daniel A. Smith
Darren Magda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmacyclics LLC
University of Texas System
Original Assignee
Pharmacyclics LLC
University of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacyclics LLC, University of Texas System filed Critical Pharmacyclics LLC
Publication of EP0758250A1 publication Critical patent/EP0758250A1/fr
Withdrawn legal-status Critical Current

Links

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    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A61K47/546—Porphyrines; Porphyrine with an expanded ring system, e.g. texaphyrine
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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    • B01J31/1805—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms the ligands containing nitrogen
    • B01J31/181—Cyclic ligands, including e.g. non-condensed polycyclic ligands, comprising at least one complexing nitrogen atom as ring member, e.g. pyridine
    • B01J31/1815—Cyclic ligands, including e.g. non-condensed polycyclic ligands, comprising at least one complexing nitrogen atom as ring member, e.g. pyridine with more than one complexing nitrogen atom, e.g. bipyridyl, 2-aminopyridine
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Definitions

  • the present invention relates to the field of macrocyclic expanded porphyrins and, more particularly, concerns texaphyrins and texaphyrin derivatives that are conjugated to a polymer or solid matrix to form a polymer- or matrix-supported texaphyrin.
  • Expanded porphyrins are large pyrrole-containing macrocyclic analogues of the porphyrins.
  • a number of expanded porphyrin systems are now known.
  • Those fully conjugated examples that contain more than four pyrrolic subunits include the smaragdyrins, sapphyrins, pentaphyrins, hexaphyrins, and superphthalocyanines.
  • a class of expanded porphyrins was developed based on the Schiff base condensation between a diformyltripyrrane and an aromatic 1,2-diamine 1 .
  • This class of expanded porphyrins known as the texaphyrins, describes an aromatic 227r benzannulene containing both 18- and 22-7r-electron delocalization pathways 1 .
  • the stabilization gained when the texaphyrin macrocycle becomes aromatic allows for considerable chemistry that is not possible in other pyrrole based macrocyclic systems.
  • texaphyrins previously described, however, are known only to be of the character such that they are monomers and aggregated monomers in solution. Thus, even though the texaphyrins possess absolutely unique properties, their uses have previously been limited to homogeneous systems, not allowing for the facile separation of the texaphyrin following the procedure. This deficiency limited the utility of texaphyrins to potential applications where the texaphyrin compound could be removed readily following the procedure in which it was used.
  • Immobilized texaphyrin systems would be especially useful if they could be made to retain the enhanced stability found when a texaphyrin is complexed to a lanthanide metal. Indeed, there is a significant need in the art for such a system as there is currently no means for effecting the stable complexation of lanthanides on a solid-support. The development of such a system would allow for heterogeneous catalysis, where it is important for the substrate to remain distinct from the metal complex. The potential for subjecting solid-supported texaphyrins to harsh conditions, such as elevated temperatures and organic solvents, without breakdown of the catalyst would also be significantly increased. The development of immobilized texaphyrins could also prove to be useful in other areas, such as in the catalysis of certain hydrolytic reactions and the concomitant separation or purification of the individual products.
  • the present invention addresses the above and several other shortcomings in the prior art through the synthesis of various matrix-supported expanded porphyrin compositions and devices.
  • the invention provides novel matrix-supported texaphyrins, including texaphyrin-containing chromatographic supports and devices, including catheters. Methods of making such matrix-supported texaphyrins are also disclosed, as are methods of using these compositions and devices, for example, in the separation and purification of anions or neutral molecular species, in the hydrolysis of molecules containing phosphate esters, as catalysts for hydrogenation and polymerization reactions and in photodynamic therapy (PDT) and magnetic resonance imaging enhancement (MRI) .
  • PDT photodynamic therapy
  • MRI magnetic resonance imaging enhancement
  • texaphyrin compounds linked to solid-support systems represents a considerable advantage, not only in terms of lanthanide complexation, but also as it allows binding of other metals and anions under conditions of site isolation.
  • This provides an important advantage in applications involving chromatography, such as the separation of molecular species or in the hydrolysis of compounds and separation of products, in a variety of catalytic mechanisms, including racemate resolution, and in MRI and PDT.
  • the invention therefore encompasses new texaphyrin derivatives, conjugates and polymers thereof, as formulated into matrix-supported texaphyrins.
  • matrix-supported texaphyrins that include one or more texaphyrin monomers, texaphyrin derivatives or conjugates or polymers thereof, whether or not the matrix-supported material also contains other groups, such as additional functional groups, or even sapphyrins.
  • a texaphyrin is defined as a class of expanded porphyrins that is based on the Schiff base condensation between a diformytripyrrole and an o- phenylenediamine.
  • the synthesis of texaphyrins depends on the simple, high yielding synthesis of tripyrroles. Following the synthesis of the tripyrrole is an acid- catalyzed condensation between a diformyltripyrrole and an o-phenylenediamine derivative. This results in the production of tripyrrole-containing Schiff base macrocycles referred to as reduced or sp 3 texaphyrins. Subsequent 4-electron oxidation either in the presence or absence of a metal cation gives rise to the final aromatic, or sp 2 , form of the texaphyrin.
  • Any texaphyrin, or derivative thereof, may be linked to a solid-support in accordance with the present invention.
  • Structure I sets forth the general structure for a texaphyrin macrocycle.
  • Texaphyrins may have a variety of substituents at the various R positions, as exemplified by hydrogen, halide, hydroxyl, alkyl, alkene, alkyne, aryl, haloalkyl, nitro, for yl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, glycol, polyglycol, thiol, thioalkyl, aminoalkyl, alkoxyalkyl, aryloxyalkyl, alkyloxycarbonyl, aryloxycarbonyl, aldehyde, ether, ketone, carboxylic acid, saccharide, phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate substituted alkyl, sulfate substituted alkyl, carboxy, carboxyalkyl, carboxyamidealkyl, a nucleobase, modified nucleobase, oligonucleotide
  • M is hydrogen or a metal cation.
  • Structures II and III show texaphyrins bonded to a solid- support (SS) .
  • the texaphyrin R groups and M may be those described for structure I.
  • X represents the group of the texaphyrin for use in bonding
  • Y represents the group of the matrix solid-support (SS) for use in bonding; XY thus represents the linker between a texaphyrin and a matrix suppor .
  • the matrix-supported texaphyrins of the present invention are useful for ester bond hydrolysis, heterogeneous catalysis, anion binding, separation of molecules such as nucleotides and oligonucleotides, magnetic resonance imaging, photodynamic therapy, or DNA photocleavage, for example.
  • Matrix-supported texaphyrins are further useful as a medical device, such as a catheter, implant, interface or artificial joint.
  • a further aspect of the present invention involves the realization that novel expanded porphyrins, and particularly, texaphyrin structures may be prepared through the construction of polymers and oligomers, generically referred to as "multimers.”
  • multimers is intended to refer to any compound that includes at least two texaphyrin macrocycles joined covalently.
  • oligomer and polymer are generally understood to be overlapping in terms of defining a given length.
  • a texaphyrin “oligomer” or “oligotexaphyrin” refers to structures having about 2 or 3, and more preferably, about 10, 15, 20 or 30 texaphyrin units per molecule, up to and including about 40 or 50 units.
  • “polymers” and “polytexaphyrin” are texaphyrin-containing structures that generally have upwards of about 40 or 50 texaphyrin units, up to an including about 80, 100 or 150 texaphyrin units, or even up to about 200 texaphyrin units or even more.
  • the texaphyrin oligomers and polymers of the invention also include texaphyrin dimers and trimers.
  • n the number of individual texaphyrin derivative units, may be about 5, 10, 20, 30 or 40, for texaphyrin oligomers, and about 50, 75, 100, 150 or about 200 or so for texaphyrin polymers.
  • the invention concerns compositions that are composed of a texaphyrin derivative in accordance with any one of the embodiments discussed above, particularly the polymer-supported texaphyrin, complexed to a second compound.
  • the invention concerns a method for forming a complex between a texaphyrin derivative and a second compound, wherein the method involves preparing a texaphyrin derivative as described above, including the polymer-supported forms such as silica-, Merrifield resin-, glass-, plastic- and organic polymer-supported texaphyrins; obtaining the second compound; and contacting the texaphyrin derivative with the second compound under conditions effective to allow the formation of a complex between the texaphyrin derivative and the second compound.
  • Structure I provides a texaphyrin having substituent groups, ⁇ R ⁇ *
  • texaphyrin derivatives and their metal complexes have been prepared, for example, as described in U.S. Patents 4,935,498, 5,162,509, 5,252,720, 5,292,414, 5,272,142, 5,256,399 and 5,369,101; PCT publications WO 94/09003 and WO/94,29316 and Application Serial Nos.
  • M may be hydrogen, a divalent metal cation or a trivalent metal cation; and R ⁇ -R 4 , R 7 and R 8 may be separately and independently hydrogen, halide, hydroxyl, alkyl, alkene, alkyne, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, glycol, polyglycol, thiol, thioalkyl, aminoalkyl, alkoxyalkyl, aryloxyalkyl, alkyloxycarbonyl, aryloxycarbonyl, aldehyde, ether, ketone, carboxylic acid, saccharide, phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate substituted alkyl, sulfate substituted alkyl, carboxyamidealkyl, a nucleic acid, phosphate, phosphonate,
  • M may be a divalent metallic cation, such as Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II), Sm(II),
  • M may be a trivalent metal cation, such as Mn(III), Co(III), Ni(III), Fe(III), Ho(III), Ce(III), Y(III), In(III), Pr(III), Nd(III), Sm(III), Sc(III), Eu(III), Gd(III), Tb(III), Dy(III), Er(III), Tm(III), Yb(III), Lu(III), La(III), or U(III) ; in which case, "A” would be 2.
  • Representative paramagnetic metal cations are Sm(III) , Eu(III), Gd(III), and Dy(III) .
  • diamagnetic metal cations are La(III), Lu(III), and Y(III) .
  • R , R 2 , R 3 , R and R 5 may be attached via a carbon-carbon or a carbon-oxygen bond. Also, where oxyhydroxyalkyl groups are present, they may be further substituted, bearing a substituent in lieu of hydrogen of the hydroxyl substituent. Additionally, where carboxyamidealkyl groups are present, they may have a secondary or tertiary amide linkage.
  • the aryl group may be phenyl, or a phenyl having a nitro, carboxy, sulfonic acid, hydroxy, oxyalkyl or a halide substituent.
  • Oxyhydroxyalkyls may be alkyls having independently hydroxy substituents and ether branches.
  • Carboxyalkyl groups may be alkyls having a carboxyl substituted ether, an amide substituted ether or a tertiary amide removed from an ether. Couples may be amide, thiol, thioether or ether covalent bonds.
  • alkanes useful as alkyl group substituents of the present invention include methane, ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with methane, ethane and propane being preferred.
  • alkenes useful as alkenyl group substituents include ethene, straight-chain, branched or cyclic isomers of propene, butene, pentene, hexene, heptene, octene, nonene and decene, with ethene and propene being preferred.
  • alkynes useful as alkynyl group substituents include ethyne, straight-chain, branched or cyclic isomers of propyne, butyne, pentyne, hexyne, heptyne, octyne, nonyne and decyne, with ethyne and propyne being preferred.
  • substituted alkyls include alkyls substituted by two or more functional groups as described herein.
  • halide substituents chloride, bromide, fluoride and iodide are contemplated in the practice of this invention with the exception of iodide for R 6 and R g .
  • R 6 and R 9 may have chloride, bromide or fluoride substituents.
  • Representative examples of haloalkyls used in this invention include halides of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane and decane, with halides, preferably chlorides or bromides, of methane, ethane and propane being preferred.
  • hydroxyalkyls include alcohols of methane, ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with alcohols of methane, ethane or propane being preferred.
  • Hydroxyalkyl is meant to include glycols and polyglycols; diols of ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with diols of ethane or propane being preferred; polyethylene glycol, polypropylene glycol and polybutylene glycol as well as polyalkylene glycols containing combinations of ethylene, propylene and butylene.
  • oxyalkyls include the alkyl groups as herein described having ether linkages.
  • the number of repeating oxyalkyls within a substituent may be up to 100, preferably is from 1-10, and more preferably, is 2-3.
  • thioalkyls include thiols of ethane, thiols of straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with thiols of ethane (ethanethiol, C 2 H 5 SH) or propane (propanethiol, C 3 H 7 SH) being preferred.
  • Sulfate substituted alkyls include alkyls as described above substituted by one or more sulfate groups, a representative example of which is diethyl sulfate ( (C 2 H 5 ) 2 S0 4 ) ; they also include simple anionic sulfate or sulfonate substituents such as -C 2 H 5 S0 3 .
  • Representative examples of phosphates include phosphate or polyphosphate groups.
  • Representative examples of phosphate substituted alkyls include alkyls as described above substituted by one or more phosphate or polyphosphate groups.
  • Representative examples of phosphonate substituted alkyls include alkyls as described above substituted by one or more phosphonate groups.
  • carboxy groups include carboxylic acids of the alkyls described above as well as aryl carboxylic acids such as benzoic acid.
  • carboxyamides include primary carboxyamides (CONH 2 ) , secondary (CONHR') and tertiary (CONR'R'') carboxyamides where each of R # and R # ' is a functional group as described herein.
  • useful amines include a primary, secondary or tertiary amine of an alkyl as described hereinabove.
  • oligonucleotides include nucleotides, oligonucleotides and polynucleotides primarily composed of adenine, cytosine, guanine, thymine or uracil bases. It is understood that the term nucleotide as used herein refers to both naturally- occurring and synthetic nucleotides, poly- and oligonucleotides and to analogs and derivatives thereof such as methylphosphonates, phosphotriesters, phosphorothioates and phosphoramidates.
  • useful steroids include any of the steroid hormones of the following five categories: progestins (e.g.
  • progesterone glucocorticoids (e.g., cortisol) , mineralocorticoids (e.g., aldosterone) , androgens (e.g., testosterone) and estrogens (e.g., estradiol) .
  • glucocorticoids e.g., cortisol
  • mineralocorticoids e.g., aldosterone
  • androgens e.g., testosterone
  • estrogens e.g., estradiol
  • useful amino acids of peptides or polypeptides include amino acids with simple aliphatic side chains (e.g., glycine, alanine, valine, leucine, and isoleucine) , amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine, and histidine) , amino acids with oxygen and sulfur-containing side chains (e.g., serine, threonine, methionine, and cysteine) , amino acids with side chains containing carboxylic acid or 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 arginine) , and proline.
  • amino acids with simple aliphatic side chains e.g., glycine, alanine, valine, leucine, and isoleucine
  • amino acids with aromatic side chains
  • useful peptides include any of both naturally occurring and synthetic di-, tri-, tetra-, pentapeptides or longer peptides derived from any of the above described amino acids (e.g., endorphin, enkephalin, epidermal growth factor, poly-L-lysine, or a hormone) .
  • useful polypeptides include both naturally occurring and synthetic polypeptides (e.g., insulin, ribonuclease, and endorphins) derived from the above described amino acids and peptides.
  • Hydroxyalkyl means alkyl groups having hydroxyl groups attached.
  • Oxyalkyl means alkyl groups attached to an oxygen.
  • Oxyhydroxyalkyl means alkyl groups having ether or ester linkages, hydroxyl groups, substituted hydroxyl groups, carboxyl groups, substituted carboxyl groups or the like.
  • Saccharide includes oxidized, reduced or substituted saccharide; hexoses such as D-glucose, D-mannose or D-galactose; pentoses such as D-ribulose or D-fructose; disaccharides such as sucrose, lactose, or maltose; derivatives such as acetals, amines, and phosphorylated sugars; oligosaccharides, as well as open chain forms of various sugars, and the like.
  • amine-derivatized sugars are galactosamine, glucosamine, sialic acid and D-glucamine derivatives such as 1-amino-1-deoxysorbitol.
  • Carboxyamidealkyl means alkyl groups with hydroxyl groups, secondary or tertiary amide linkages or the like.
  • Carboxyalkyl means alkyl groups having hydroxyl groups, carboxyl or amide substituted ethers, ester linkages, tertiary amide linkages removed from the ether or the like.
  • oxyhydroxyalkyl may be alkyl having independently hydroxy substituents and ether branches or may be C (n _ ⁇ ) H ⁇ 2n+1j _ 2 ⁇ j O ⁇ O or OC (n _ x ) H ( (2 n + i ) - 2 x)°x° y where n is a positive integer from 1 to 10, x is zero or a positive integer less than or equal to n, and y is zero or a positive integer less than or equal to ( (2n+l) -2x) .
  • the oxyhydroxyalkyl or saccharide may be C n H, (2n+1) _ q) O y R a q , OC rP i Un+D - O y --- or (CH 2 ) n C0 2 R wnere is a positive integer from 1 to 10, y is zero or a positive integer less than ( (2n+l) -q) , q is zero or a positive integer less than or equal to 2n+l, R a is independently H, alkyl, hydroxyalkyl, saccharide, C (m . w) H ( ⁇ 2m+1) - 2w) 0 w 0 z'
  • Carboxyamidealkyl may be alkyl having secondary or tertiary amide linkages or (CH 2 ) n CONHR a , 0(CH 2 ) n CONHR a , (CH 2 ) n CON(R a ) 2 , or 0(CH 2 ) n CON(R a ) 2 where n is a positive integer from 1 to 10, R a is independently H, alkyl, hydroxyalkyl, saccharide, C (m _ w) H ⁇ ⁇ 2m+1) _ 2w) O w O z ,
  • the carboxyalkyl may be alkyl having a carboxyl substituted ether, an amide substituted ether or a tertiary amide removed from an ether or C n H, (2n+1) _ q) O y R c q or OC n H ((2n+1) ) O y R c q where n is a positive integer from 1 to 10; y is zero or a positive integer less than ( (2n+l) - q) , q is zero or a positive integer less than or equal to 2n+l, R c is (CH 2 ) n C0 2 R d , (CH 2 ) n CONHR d or (CH 2 ) n CON(R d ) 2 where n is a positive integer from 1 to 10; R d is independently H, alkyl, hydroxyalkyl, saccharide, C m _ w) H ((2m + l)-2 W ) 0 w 0 z
  • m is a positive integer from 1 to 10
  • w is zero or a positive integer less than or equal to m
  • z is zero or a positive integer less than or equal to ((2m+l)-2w)
  • R is H, alkyl, hydroxyalkyl, or C m H ((2m+1) _ rj O z R b r
  • m is a positive integer from 1 to 10
  • z is zero or a positive integer less than ( (2m+l) -r)
  • r is zero or a positive integer less than or equal to 2m+l
  • R b is independently H, alkyl, hydroxyalkyl, or saccharide.
  • These groups may be at any of the substituents, R 1 -R 4 , R 7 or R 8 , of the texaphyrin.
  • the X group reacts with available groups, termed Y, present on, or previously inserted into, the polymeric or solid-support matrix.
  • Y in structures II and III may thus be [- (CH 2 ) n -] -OH where n is an integer from 0 to 20, NH 2 , C0 2 H, Cl, Br, I, NCO, NCS, oxirane, C ⁇ CH, MgCl, ZnCl, Li, a nucleotide or an oligonucleotide.
  • the solid-support may be silica, silica gel, amino-functionalized silica gel, alumina, clay, zeolite, glass, controlled pore glass, montmorillonite, polystyrene, polyethylene, polyacrylamide, polypropylene, polyamide, Merrifield resin, sepharose, agarose, polystyrene, polydivinylbenzene, cellulose, alginic acid, chitosan, chitin, polystyrene-benzhydrylamine resin, an acrylic ester polymer, a lactic acid polymer, polyurethane, polyvinylchloride, nylon, latex, silicone rubber, a halogenated polyethylene, an organosilicone, a biocompatible ceramic, bioglass or sintered hydroxyapatite.
  • Structures IV and V show examples of structures II and III, respectively, representing solid-supported texaphyrins linked to silica gel.
  • the texaphyrin units for use in conjugating to a solid-support may also be dimers, trimers, oligomers or polymers of texaphyrins or texaphyrin derivatives alone; or may be dimers, trimers, oligomers or polymers of texaphyrins or texaphyrin derivatives in combination with other expanded porphyrins, such as sapphyrins or rubyrins.
  • the texaphyrins may also be conjugated to other units, such as alkyl chains or nucleobases, prior to linking to a polymer or solid-support.
  • texaphyrin-metal complex refers to an aromatic pentadentate expanded porphyrin analog metal complex, that may have various appended functional groups.
  • metals may be complexed with a texaphyrin. These include, for example, divalent or trivalent metal cations of the lanthanide group or Lewis acidic cations.
  • Examples of these include all of the lanthanide group with the exception of Pm(III) , as represented by La(III), Nd(III), Sm(III) , Sm(II), Gd(III), Tm(III) , Lu(III) , Eu(III) and Dy(III) ; and also Y(III) , In(III) , Ce(III) and Sc (III) .
  • divalent and trivalent metallic cations that may be bound to a matrix-supported texaphyrin of the invention include Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II), and Mn(III), Co(III), Ni(III), Fe(III), Cu(II), Ho(III), Pr(III), Tb(III), Er(III), Yb(III), with even V(III) , Cr(II) , and Cr(III) being possibilities for binding to texaphyrins.
  • metal ion for complexing to a texaphyrin will generally be dependent upon the use or uses intended for the matrix-supported texaphyrin.
  • metal ions that have catalytic activity for ester bond hydrolysis such as Eu(III) or Dy(III) would be used to create a matrix-supported metallotexaphyrin for use in RNA hydrolytic cleavage.
  • Metal ions that have catalytic activity for hydrogenation and polymerization reactions may be used to construct a matrix-supported metallotexaphyrin for use in heterogeneous catalysis.
  • the metals e.g., by changing from Gd(III) to Lu(III) or Ce(III)
  • the texaphyrin compounds can be modified so as to have enhanced selectivity for various different anions such as phosphates and carboxylates.
  • Paramagnetic species such as Gd(III), Cu(III), Sm(III), Eu(III), Dy(III), Ni(III), Co(II), Co(III),
  • Fe(II) and Fe(III), and most preferably, Gd(III), that have utility in magnetic resonance imaging protocols are also useful metals for complexing with matrix-supported texaphyrins.
  • diamagnetic species such as La(III), Y(III) or Lu(III)
  • matrix-supported texaphyrins would then be useful in photodynamic therapy.
  • Lu(II) or La(III) derivatives would thus be preferred in applications, such as in vitro purging of virus and cancer cells from blood or other bodily fluids, where the photodynamic production of singlet oxygen is of predicative importance.
  • 90 ⁇ 3 + anc j n i n 3 + deriv tives would be of preference in radiotherapeutic and diagnostic applications.
  • texaphyrins are photosensitive and are useful for photodynamic therapy and other applications where the generation of toxic oxygen products, such as singlet oxygen, is desired.
  • Nonmetallated systems would be further useful for the chromatography, capture and/or removal of metals, both on an analytical scale and an industrial one.
  • the texaphyrin may be conjugated to the matrix at virtually any R position and using a wide range of R groups.
  • Y in structures II and III may thus be [- (CH 2 ) n -] -OH, NH 2 , C0 2 H, Cl, Br, I, NCO, NCS, oxirane, C ⁇ CH, MgCl, ZnCl, Li, a nucleotide or an oligonucleotide.
  • a polymeric or solid-support matrix comprises, or may be derivatized to comprise, a group that permits the formation of a covalent bond with a texaphyrin substituent.
  • Groups such as aryl, silyl, siloxy, aminoaryl, amino, amidoaryl and silyloxy are examples of these.
  • the linker between a texaphyrin and a matrix may also be described using the formula: T- (CH 2 ) n -A- (CH 2 ) m -B, wherein T is any texaphyrin macrocycle or derivative or conjugate thereof; B is the polymeric matrix or solid-support matrix; and n and m are zero or integers of less than or equal to 20, or more preferably, of less than or equal to 10.
  • A may be alkyl, aryl, oxy, sulfide, amide, carbonyl, alkenyl, alkynyl, halide, alkylhalide, arylhalide, arenyl, hydroxyalkyl, glycol, polyglycol, thiol, alkylthiol, substituted alkyl, phosphate, phosphonate, sulfate, phosphate substituted alkyl or aryl, phosphonate substituted alkyl or aryl, sulfate substituted alkyl or aryl, carboxy, carboxyamide, ester, thiol-substituted carboxyamide, or derivatized carboxyamide.
  • the functionalization of a polymeric matrix or solid-support matrix may take any one of many forms, so long as the result is to derivatize the polymer or support so that a texaphyrin moiety may be attached or bonded to the support.
  • the polymeric or solid-support matrix will thus comprise, or will be derivatized to comprise, an aryl, silyl, siloxy, aminoaryl, amino, amidoaryl or silyloxy group, which group will form as a linking unit (linker) between the solid-support and the texaphyrin.
  • linkages between the texaphyrin and the polymeric or solid-support matrix include: - (CH 2 ) 3 - (CH 2 ) 2 -Si(R) 2 -0-; -(CH 2 ) 3 -
  • Si(R) 2 0- and - (CH 2 ) 2 -Si- (CH 2 ) 2 0-, where R may be siloxy, alkyl, hydroxyl, alkoxy, or oxygen, for example.
  • polymeric or solid-support matrices may be linked to a texaphyrin as disclosed herein. Although there is overlap in terminology, the polymers or matrices may be considered as inorganic polymers or matrices, organic polymers, copolymers and biocompatible polymers, all of which can be functionalized by those skilled in the art of chemical synthesis.
  • inorganic polymers or matrices include, for example, silica, silica gel, amino-functionalized silica gel, alumina, clay, zeolite, glass, controlled pore glass and montmorillonite, along with certain biocompatible materials.
  • organic polymers include polystyrene, polyethylene, polyacrylamide, polypropylene, polyamides, Merrifield resin, sepharose, agarose; with copolymers being represented by polystyrene and polydivinylbenzene.
  • Polysaccharides are another group of organic polymers. These include cellulose and alginic acid and amino-polysaccharides, such as chitosan and chitin. In addition to amino-functionalized natural polymers, man- made amino-functionalized organic polymers are also contemplated, such as polystyrene-benzhydrylamine resin. In addition to certain natural organic polymers, other biocompatible polymers include, for example, polyurethane, polyvinylchloride, nylon, latex, silicone rubber, halogenated polyethylenes, such as polytetrafluoroethylene (PTFE) and other Teflon ® materials, organosilicones, e.g., Silastic ® materials, and even biocompatible ceramics.
  • PTFE polytetrafluoroethylene
  • matrices for use in medical compositions and devices these should be “biocompatible”. This means that the matrix has all the features commonly associated with medical uses, in that it is in a form that does not produce an adverse, allergic or other untoward reaction when administered to an animal or human subject.
  • matrix material will differ according to the particular circumstances and the site of the body into which the device is to be inserted. Physical and chemical characteristics, such as, e.g., biocompatibility, biodegradability, strength, rigidity, interface properties and even cosmetic appearance may be considered in choosing a matrix, as is well known to those of skill in the art.
  • Non-biodegradable matrices will most often be employed in accordance with the polymer-supported texaphyrins, such as, sintered hydroxyapatite, bioglass, aluminates, or other bioceramic materials. Ceramic delivery systems are described in U.S. Patent 4,596,574, incorporated herein by reference. Polymeric matrices that may also be employed include acrylic ester polymers and lactic acid polymers, as disclosed in U.S. Patents 4,526,909, and 4,563,489, respectively, each incorporated herein by reference.
  • the polymeric or solid-support matrix that forms the supporting part of the matrix-supported texaphyrin will be chosen according to the intended function and use of the resultant composition.
  • silica gel may be used to prepare bonded, texaphyrin-substituted silica gels for formulation into columns for use in medium to high pressure applications, such as in analytical and preparative HPLC separation technology.
  • organic polymer-based texaphyrins may be prepared, such as texaphyrin-substituted Merrifield resins.
  • a glass support such as a glass capillary tube, may be employed to prepare a glass-supported texaphyrin for use, e.g., in analytical capillary electrophoresis (CE) .
  • CE analytical capillary electrophoresis
  • compositions of the invention include medical devices that comprise a texaphyrin linked to a surface of the device, for example, a catheter in which one or more texaphyrins are linked to a surface of the catheter; an orthopedic implant or interface or an artificial joint.
  • a texaphyrin linked to a surface of the device for example, a catheter in which one or more texaphyrins are linked to a surface of the catheter; an orthopedic implant or interface or an artificial joint.
  • implants themselves and functional parts of implants, such as, e.g., surgical screws, pins, and the like are encompassed.
  • a catheter, implant, or a portion thereof, such as a screw will be coated with a metallotexaphyrin complexed to a paramagnetic metal ion, such as Cu(III), Ni(III), Co(II), Co(III), Fe(II) or Fe(III) , and most preferably, Gd(III), prior to implantation.
  • a metallotexaphyrin complexed to a paramagnetic metal ion such as Cu(III), Ni(III), Co(II), Co(III), Fe(II) or Fe(III) , and most preferably, Gd(III), prior to implantation.
  • a second example contemplated concerns the use of matrix-supported texaphyrin constructs that also comprise nucleobase structures and thus allow separation of nucleobase- containing compounds on the basis of specific base- pairing interactions.
  • further modified matrix-supported texaphyrins may be constructed by modifying either the appended texaphyrin moiety or the polymeric or solid-support matrix itself.
  • a texaphyrin-nucleobase conjugate may be used in the initial synthesis, or alternatively, the nucleobase units may be later appended onto available groups of the texaphyrin or onto available groups of the solid-support itself.
  • long chain alkyl groups it is contemplated that such groups will generally be introduced onto the surface of the polymeric or solid-support matrix, although the invention is not limited solely to this mode of addition.
  • the range of matrix-supported texaphyrin-nucleobase constructs contemplated includes a wide variety of nucleobase compounds.
  • the choice of compound will be tailored to suit the intended function of the solid- support, such as, to bind to a specific nucleobase- containing compound which one desires to purify, remove, or otherwise separate from a mixture of compounds.
  • the nucleobase-containing group may therefore comprise any purine or pyrimidine base including adenine, cytosine, guanine, thymidine, uridine and inosine, or any analog or derivative thereof, including antimetabolite nucleobases, nucleobase derivatives shown in Table 2, and even protected nucleobases.
  • the texaphyrin-nucleobase solid-support matrices may also have appended nucleosides, nucleotides and oligonucleotides, for example, oligonucleotides comprising between two and about 10 nucleobase units. Constructs bearing a selected nucleotide or oligonucleotide may be formulated into columns, filters or other solid materials such as capillary tubes, and used to selectively bind compounds which include the complementary nucleotide or oligo or poly-nucleotides containing substantially complementary sequences.
  • a substantially complementary sequence is one which the nucleotides generally base pair with the complementary nucleotide and in which there are very few base pair mismatches.
  • Matrix-supported texaphyrins of the second and third generation may also comprise functional groups designed to alter the chemical capabilities of the metallotexaphyrins.
  • the texaphyrin may include an imidazole ring or an arginine residue.
  • the texaphyrin may also have an attached chiral group, for example: a D-sugar unit, such as D-glucopyranosyl; an L-sugar unit; an L-amino acid, such as L-alanine or L-phenylalanine; an oligopeptide; an oligosaccharide; or a chiral binaphthyl system.
  • the present invention also encompasses other matrix-supported expanded porphyrins, including sapphyrins and rubyrins, that have been joined to a polymeric or solid-support matrix.
  • the use of an expanded porphyrin capable of binding anions, and preferably, those capable of phosphate or nitrate chelation, is particularly contemplated.
  • the use of expanded porphyrins of the rubyrin class, and particularly of the sapphyrin class is envisioned.
  • the present invention also provides methods for using the matrix-supported texaphyrins and expanded porphyrins described above in separating a first molecule from a mixture of at least two molecules.
  • the term "molecule" is being used for simplicity to refer to both molecular and atomic structures and thus encompasses species such as metal cations like those of the trivalent lanthanide series and atomic anions such as Cl ⁇ , Br " , and I " .
  • the columns, capillary tubes, filters, devices and the like, of the invention are contemplated to be of most use in separating neutral or negatively- charged entities that would be bound as ligands to the texaphyrin metal centers.
  • Such species could be neutral materials containing, e.g., hydroxyls; or anionic materials, such as those bearing a phosphate, phosphonate, phosphate ester, arsenate, arsenate ester, carboxylate, nitrate, sulfate, sulfonate or sugar moiety within their structure.
  • the matrix-supported texaphyrin constructs will generally be of most use for separating oligonucleotides such as RNA and DNA that contain phosphate esters.
  • these same solid-supports could be used to effect catalytic hydrolysis of the phosphate ester bonds, as described in the present detailed examples.
  • a first molecule such as an RNA fragment of a given length from a mixture containing at least one other oligonucleotide of similar but not identical composition in accordance with the invention
  • a matrix support bearing a texaphyrin with the mixture, thereby separating the first molecule.
  • the contacting may, generally, take the form of passing a solution containing the mixture to be separated, or purified, over a column to which texaphyrins are appended.
  • the separation process generally entails binding the mixture containing the molecules or species to be separated to a matrix-supported texaphyrin, such as a column, capillary tube, filter, filter cartridge or such like, and then, subsequently, removing, or eluting, the bound species in such a way as to result in the formation of distinct fractions containing molecules which have been separated from each other.
  • a matrix-supported texaphyrin such as a column, capillary tube, filter, filter cartridge or such like
  • chromatographic or electrophoretic separation methods The methods for separating or purifying anionic, cationic, or even neutral species by contacting solutions of various mixtures with a functionalized solid-support are generally encompassed by the terms "chromatographic or electrophoretic separation methods".
  • the chromatographic methods are those using columns, such as HPLC columns, whereas the electrophoretic separation methods are exemplified by those using capillary tubes.
  • Various chromatographic and electrophoretic separation techniques are well known in the art, and any such method may be employed in connection with the invention simply by preparing a polymer-supported texaphyrin and packaging or formulating it to give a device to be used as the central component of the separation technique.
  • anionic, cationic or even neutral species from a mixture in accordance with the invention, it is contemplated that one would generally first formulate the mixture to be separated into a solution. One would then contact the matrix-supported texaphyrin with the solution under conditions effective, and for a time period sufficient, to allow binding of the anionic, cationic or neutral species to the solid-support. This is straightforward and may be achieved simply by passing the solution over the solid material with or without the use of pressure or an electrostatic field.
  • the anionic or neutral species will, in the simplest case, bind specifically to the metal center of the texaphyrin moiety.
  • matrix-supported texaphyrins bear other groups, such as long chain alkyl groups, nucleobases, or aromatic residues
  • other portions of the contemplated substrates such as hydrophobic portions or purine and pyrimidine nucleobase substituents in this case, e.g., of oligonucleotides will also likely specifically bind to these additional functional groups or interact with the texaphyrin ring.
  • Suitable conditions effective to allow binding of the species to the solid-support will be chosen based upon considerations such as the number and type of the particular species to be separated, the purity of the resultant compositions desired, the particular type of texaphyrin and its complexed metal ion, other functional groups appended to the texaphyrin or to the solid-support matrix, and the like.
  • the solid-support may be washed with the same or other buffers of chosen stringency and for varying periods of time to remove any non-specifically bound species from the solid-support.
  • the matrix-supported texaphyrin with the bound species would next generally be treated to remove the bound species that may then be collected in a more separate and purified form than when applied, for example, collected in distinct fractions.
  • This process will generally be achieved by washing the solid-support with a second solution effective to detach the bound anionic or neutral species, i.e., a solution effective to disrupt the specific binding between the species and the texaphyrin, metal center or between it and other functional groups incorporated within the texaphyrin structure or the support.
  • a second solution effective to detach the bound anionic or neutral species i.e., a solution effective to disrupt the specific binding between the species and the texaphyrin, metal center or between it and other functional groups incorporated within the texaphyrin structure or the support.
  • Such separations in the case of chromatographic applications would be useful both for analysis and purification procedures. In the case of capillary electrophoresis, however, they would be likely to be useful only for analysis.
  • the second solution will be distinct from that first used and, again, will be chosen based upon the particular application. It may, for example, have a different ionic strength, hydrophobicity or pH compared to the first solution and may contain different concentrations of salt or other chaotropic agents as desired.
  • This removal process may be termed "elution" and the eluted material will generally be collected in relatively small fractions, such as in 1 to 2 ml fractions, and preferably in fractions of about 1 ml or smaller, so that the fractions contain one or more detached species that have been separated or purified away from the total number of species in the starting composition. Alternatively, for analysis applications, the fractions could be much smaller, i.e.
  • any of the matrix-supported texaphyrin derivatives described above may be used as a separating apparatus in this regard, as may supports comprising a sapphyrin or rubyrin derivative, and those including nucleobase conjugates, long chain alkyl groups and oligonucleotides.
  • supports comprising a sapphyrin or rubyrin derivative, and those including nucleobase conjugates, long chain alkyl groups and oligonucleotides.
  • the separating apparatus may use, for example, silica gel, Merrifield resins, glass, agarose or sepharose, polyacrylamide or another type of plastic as the support matrices and may be prepared in virtually any solid form including columns, filters, cartridges, tubes and thin layers.
  • silica gel for example, silica gel, Merrifield resins, glass, agarose or sepharose, polyacrylamide or another type of plastic
  • polyacrylamide or another type of plastic may be prepared in virtually any solid form including columns, filters, cartridges, tubes and thin layers.
  • Their use in analytical and preparative modes in both research and clinical laboratories is envisioned, as is their use in medical and veterinary procedures, including various diagnostic embodiments.
  • novel matrix-supported expanded porphyrins will be found to be particularly useful in separating compounds which have nitrate or phosphate groups or phosphate esters within their structure.
  • This includes separating or purifying purine- and pyrimidine-containing compounds, including nucleotides, oligonucleotides, gene fragments, nucleobase analogs, and derivatives such as antimetabolite purines and pyrimidines, e.g., AZT phosphate, dideoxycytidine phosphate, and other prodrugs used in the treatment of viral infections including HIV.
  • the use of the methods and compositions of the invention in clinical analyses to distinguish active phosphorylated nucleotide analogs from naturally occurring phosphorylated products, such as AMP or GMP, is a further particular utility envisioned by the inventors.
  • Organophosphorus compounds, nitrate esters, as well as simple inorganic nitrate, nitrite, phosphate and phosphonate anions, particularly pesticides, herbicides, fungicides, marine pollutants and even chemical warfare agents may also be separated in the manner of the invention.
  • using polymer-supported expanded porphyrin constructs is envisioned to be of use in removing various such undesirable compounds from contaminated solutions, e.g., in waste-removal and treatment regimens.
  • nucleotide mono-, di- and tri- phosphates may be advantageously separated from each other using polymer-supported texaphyrins, as may mono- nucleotides, di-nucleotides and various length oligonucleotides, such as oligonucleotide probes and primers.
  • the separation of larger polynucleotides, up to and including gene fragments, genes, and antisense constructs is also encompassed by the present invention.
  • sequence-specific purification of nucleotide- containing constructs is also envisioned, whereby specific oligonucleotides or polynucleotides, whether DNA or RNA species, may be obtained by employing second or third generation matrix-supported expanded porphyrins bearing appended oligonucleotides with specific sequences. This allows the separation of nucleotides and oligonucleotides not only on the basis of charge and length but also on the basis of nucleic acid type.
  • the present invention also provides methods for hydrolysing a phosphate ester bond. These methods generally comprise contacting a matrix-supported metal- texaphyrin (metallotexaphyrin) complex with a composition comprising one or more molecules or species that include within their structure a phosphate ester bond, in order to cleave said phosphate ester.
  • This process generally takes the form of contacting the matrix-supported texaphyrin with a solution that includes the molecule(s) to be cleaved under conditions effective, and for a time period sufficient, to allow functional contact between the metal-complexed texaphyrin and the molecule. The functional interaction thus results in cleavage of the ester linkage.
  • the use of matrix-supported texaphyrins allows facile recovery of the catalyst without the risk of substrate or product contamination by the catalyst. Flow reactions are also possible using these "heterogeneous systems”. These are therefore generally preferred over the batch processes possible with "homogeneous systems”.
  • the matrix-supported texaphyrin will be complexed to a metal ion, such as a divalent or trivalent metal cation, that has catalytic activity for ester bond hydrolysis, particularly in aqueous environments.
  • Examples of useful metals that may be complexed with a texaphyrin in this manner are La(III), Nd(III), Sm(III), Gd(III), Tm(III), Lu(III), Eu(III), Dy(III), Y(III) and In(III); with Eu(III)- and Dy(III) -complexed texaphyrins being preferred in certain embodiments as these texaphyrin- metal complexes are particularly effective in mediating RNA cleavage.
  • phosphate esters may be hydrolyzed, or cleaved, according to the methods of the present invention, including phosphate monoester, diester and even triester linkages.
  • Exemplary ester bonds that may be cleaved using matrix-supported texaphyrins are those present within physiologically important molecules, such as, nucleic acids, including, RNA from viral, bacterial and other pathogenic sources, as well as nucleic acid transcripts from oncogenes; mediators of metabolism, e.g., nucleotides such as ATP, ADP, AMP, cAMP, GTP, GDP, UDP, and cofactors such as NADH, NADPH, FAD, FADH 2 and UDP-glucose; phospholipids, such as phosphatidylcholine and phosphatidylethanolamine; other phosphate anhydrides; and a wide variety of assay substrates, such as p-nitrophenylphosphate ester.
  • the present invention also provides advantageous methods for conducting a chemical reaction catalyzed by a lanthanide metal.
  • These methods generally comprise preparing a matrix-supported metallotexaphyrin complex in which the texaphyrin is complexed to a lanthanide metal, and contacting the matrix-supported metallotexaphyrin complex with a composition comprising the substrates for the lanthanide metal-catalyzed chemical reaction.
  • Contacting would generally be with a solution that includes the substrate molecule(s) for the reaction under conditions effective, and for a time period sufficient, to allow functional contact between the metal complexed texaphyrin and the molecule(s).
  • Metallotexaphyrins complexed to La(III), Sm(III), Sm(II), Eu(III), Ce(III) or Dy(III) may be found to be particularly useful. These methods are suitable for the catalysis of reactions such as ester hydrolysis, amide hydrolysis, phosphate ester hydrolysis, acyl transfer, hydrogenation and polymerization.
  • Improved methods for MRI form another aspect of the invention may be used to locate a device or catheter within the body.
  • a matrix-supported metallotexaphyrin complex comprising a paramagnetic metal ion, such as Gd(III), Cu(II), Ni(III), Co(II), Co(III), Fe(III) or Fe(III), then generally formulate this polymer-supported texaphyrin into a device and insert the device into an animal.
  • Gd(III) is particularly preferred in such embodiments.
  • the invention provides new methods for light-induced singlet oxygen production using matrix-supported texaphyrins where the texaphyrin is either unmetallated or complexed to a diamagnetic metal.
  • Preferred diamagnetic metal cations are Lu(III) or La(III), for example.
  • Photoirradiation is used in a sufficient quantity to induce singlet oxygen production at levels that are cytotoxic to the tumor cells. This is particularly suitable for use in deleting tumor cells from the blood of patients that have, or are suspected to have, leukemia, so that the purged blood may be re-administered to the patient.
  • Another method of exploiting singlet oxygen formation using a matrix-supported texaphyrin is to deactivate a retrovirus or enveloped virus, such as HIV- 1, HIV-2, HSV, SIV or FIV.
  • a retrovirus or enveloped virus such as HIV- 1, HIV-2, HSV, SIV or FIV.
  • a matrix-supported texaphyrin or texaphyrin diamagnetic metal complex can be prepared; contact this with a composition suspected of comprising a retrovirus or enveloped virus; and then photoirradiate the composition in contact with the matrix-supported texaphyrin or texaphyrin diamagnetic metal complex to produce singlet oxygen in a quantity cytotoxic to any virus.
  • viruses may be removed from blood and blood products suspected of comprising retrovirus or enveloped virus. This is useful not only in terms of rendering blood free from virus for use in transfusions, but also for use in rendering blood products and sera free from virus for use as growth media, e.g., in monoclonal antibody generation.
  • a further use of the matrix-supported texaphyrins involves the discovery that photosensitive texaphyrins catalyze the cleavage of a polymer of deoxyribonucleic acid. Cleavage is enhanced by the presence of oxygen, indicating that singlet oxygen or another oxygen by- product is the likely toxic agent.
  • a photosensitive texaphyrin may be a diamagnetic metal texaphyrin complex or may be metal-free.
  • the interaction between a matrix-supported texaphyrin-oligonucleotide conjugate and the complementary oligonucleotide is an example of antisense technology and allows cleavage of a polymer of deoxyribonucleic acid that is in the vicinity of the specific binding.
  • the inherent biolocalization properties of texaphyrin further effect targeting of an antisense agent to lipophilic regions, especially tumors and atheroma, for example.
  • texaphyrin-oligonucleotide conjugate means that an oligonucleotide is attached to the texaphyrin in a 5' or 3' linkage or both types of linkages to allow the texaphyrin to be an internal residue in the conjugate.
  • the oligonucleotide or other site-directing molecule may be attached either directly to the texaphyrin via a linker, or a couple of variable length.
  • the texaphyrin portion of a texaphyrin metal complex-oligonucleotide conjugate is placed in the vicinity of the substrate upon binding of the oligonucleotide to the targeted nucleic acid substrate.
  • a "sapphyrin-oligonucleotide conjugate" is referred to in the same way as described above for a texaphyrin-oligonucleotide conjugate except that the texaphyrin is replaced with a sapphyrin.
  • a couple may be described as a linker, i.e., the covalent product formed by reaction of a reactive group designed to attach covalently another molecule at a distance from the texaphyrin macrocycle.
  • linkers or couples are amides, amine, thioether, ether, or phosphate covalent bonds as described in the examples for attachment of oligonucleotides.
  • oligonucleotides and other site-directing molecules are covalently bonded to the texaphyrin via a carbon-nitrogen, carbon-sulfur, or a carbon-oxygen bond.
  • the cleavage of DNA described herein is a photolytic cleavage. It is believed that the cleavage is not hydrolytic where a water molecule is added across a bond to break the bond, nor is the cleavage believed to be solely oxidative where an oxidation reaction in the absence of light causes breakage of the bond.
  • the site-specific cleavage of DNA has important ramifications in a variety of applications. Potential particular applications for this process include antisense applications; the specific cleavage and possible subsequent recombination of DNA; destruction of viral DNA; construction of probes for controlling gene expression at the cellular level and for diagnosis; and cleavage of DNA in footprinting analyses, DNA sequencing, chromosome analyses, gene isolation, recombinant DNA manipulations, mapping of large genomes and chromosomes, in chemotherapy and in site-directing mutagenesis.
  • the term "photosensitive" means that upon irradiation, texaphyrin effects either the generation of oxygen products that are cytotoxic or means that the texaphyrin is fluorescent, or both. Cytotoxic oxygen products may be singlet oxygen, hydroxyl radicals, superoxide, or hydroperoxyl radicals.
  • the texaphyrin may be a diamagnetic metal complex or a metal-free species. Diamagnetic metals would include preferably, Lu(III), La(III), In(III), Zn(II) or Cd(II). Most preferably, the diamagnetic metal is Lu(III).
  • a further embodiment of the present invention provides a method of light-induced cleavage of a polymer of deoxyribonucleic acid.
  • the method comprises the steps of contacting the polymer with a matrix-supported photosensitive texaphyrin and exposing the matrix- supported photosensitive texaphyrin to light for a time sufficient to cleave the polymer.
  • the exposing step is carried out in the presence of oxygen.
  • the texaphyrin may be a metal complex of texaphyrin, preferred metals are diamagnetic metals.
  • the polymer may be a solution or a suspension of DNA or may be cellular DNA in vi tro or in vivo. DNA is preferably cleaved over RNA.
  • Another embodiment of the present invention is a method for targeted intracellular DNA cleavage.
  • the method comprises the introduction into a cell of a matrix-supported texaphyrin coupled to an oligonucleotide having complementary binding affinity for a targeted DNA, whereby cleavage of the targeted DNA is catalyzed by the matrix-supported texaphyrin.
  • the DNA may be oncogene DNA or may be normal DNA which needs to be destroyed, for example, due to improper timing of expression.
  • the oligonucleotide coupled to the matrix-supported texaphyrin may be DNA, a DNA analog, or an RNA analog oligonucleotide.
  • the texaphyrin may be a free base texaphyrin or a metallated form of texaphyrin.
  • the metal is preferably a diamagnetic metal, most preferably Lu(III) .
  • a method for inhibiting the expression of a gene in an animal comprising the administration to the animal of a matrix-supported texaphyrin oligonucleotide-conjugate is a further embodiment of the present invention.
  • the oligonucleotide may have complementary binding affinity for regulatory regions of the gene or for regions encoding exons or introns.
  • the oligonucleotide may be complementary to either strand of the DNA or to the duplex DNA.
  • a further embodiment of the present invention is a method for inhibiting the expression of a gene in a particular tissue of an animal comprising administering to the animal a matrix-supported texaphyrin having specificity for the tissue.
  • the texaphyrin may have appended an oligonucleotide complementary to the target gene.
  • a further embodiment of the present invention is a matrix-supported texaphyrin conjugate wherein two or more separate matrix-supported texaphyrin complexes are attached to an oligonucleotide, one at the 3', one at the 5' end, and/or one or more at an internal residue.
  • the texaphyrin may be metal free or may be metallated.
  • a metal ion of each of the matrix-supported texaphyrin complexes may be the same or it may be different.
  • each of the matrix-supported texaphyrins may be different.
  • Use of a dual texaphyrin complex-conjugate should effect the cleavage of DNA with increased efficiency due to the concerted activity of the metal complexes.
  • the administration of such a matrix-supported conjugate with one texaphyrin complex having a diamagnetic metal species and the other having a paramagnetic species would allow binding, imaging, and cleavage, all effected by one conjugate.
  • binding is effected by the oligonucleotide
  • imaging is accomplished by MRI due to the presence of the paramagnetic metal ion
  • cleavage is accomplished by the photosensitive texaphyrin containing a diamagnetic metal cation. Therefore, the biodistribution and cellular penetration of the matrix-supported conjugate may be determined.
  • the matrix-supported texaphyrins may be formulated into various columns, including HPLC columns; capillary electrophoresis tubes; and various other devices such as filters and large reaction vessels.
  • the present invention generally concerns polymeric matrices or solid-supports linked to texaphyrins
  • the invention provides for the linkage of further groups to a texaphyrin.
  • groups include, for example, sugars, sugar derivatives, polysaccharides, metal chelating groups, alkylating agents, nucleobases, modified nucleobases, oligonucleotides, antibodies, hormones, steroids, steroid derivatives, amino acids, peptides, polypeptides, other texaphyrins and texaphyrin derivatives, rubyrin and rubyrin derivatives, sapphyrins and sapphyrin derivatives, polymeric sapphyrins, polymeric texaphyrins, and the like.
  • texaphyrin derivatives that are polyhydroxylated and therefore water-soluble.
  • Water soluble texaphyrins are particularly desirable where one would like to exploit the various surprising properties of these macrocycles in connection with human or animal applications.
  • the nature of the polyhydroxylation is not believed to be particularly critical to achieving water solubility of a texaphyrin derivative, so long as at least two hydroxyl groups per macrocycle are incorporated into the structure. This is similar to sapphyrins, where the present inventors have found that the introduction of at least 1 or 2 hydroxyl groups per macrocycle was sufficient to achieve some degree of sapphyrin water solubility.
  • exemplary polyhydroxylated texaphyrins are those that are modified to include structures such as hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, dihydroxyalkyl, trihydroxyalkyl, or the like, at one or more R positions of the basic structure.
  • sugars are not critical to the achievement of water solubility, and a non-exhaustive, exemplary list of sugars contemplated to be useful in this regard is set forth in Table 1.
  • any sugar or modified sugar may be employed including sugars having additional phosphate, methyl or amino groups and the like.
  • the use of both D- and L-forms, as well as the c. and ⁇ forms is also contemplated.
  • preferred sugars for use in accordance herewith will include, for example, glucose, glucosamine, galactose, galactosamine and mannose.
  • RNA cleavage using Eu(III)- texaphyrins and solid-supported Eu(III) -texaphyrins is described herein in Example 14.
  • the present invention relates to what are referred to as expanded porphyrin, and particularly, texaphyrin-nucleobase conjugates.
  • texaphyrin-nucleobase conjugate is intended to refer broadly to any conjugate formed by the covalent conjugation of any texaphyrin macrocycle to any nucleobase.
  • the texaphyrin-nucleobase conjugates may also be attached to polymeric matrices or solid-supports, such as silica gel, glass, Merrifield resins, polyacrylamide, polystyrene, sepharose, agarose, clays, zeolites, plastics, biocompatible matrices and the like; to form a chromatography column, filter or medical device.
  • polymeric matrices or solid-supports such as silica gel, glass, Merrifield resins, polyacrylamide, polystyrene, sepharose, agarose, clays, zeolites, plastics, biocompatible matrices and the like.
  • nucleobase is intended to refer broadly to any moiety that includes within its structure a purine or pyrimidine, a nucleic acid, nucleoside, nucleotide, or any derivative of any of these.
  • nucleobase includes adenine, cytosine, guanine, thymidine, uridine, inosine, or the like, bases, nucleotides or nucleosides, as well as any base, nucleotide or nucleoside derivative based upon these or related structures.
  • nucleobases are the so-called antimetabolites, which are generally based upon the purine or pyrimidine structures. These compounds typically exert their biological activity as antimetabolites through competing for enzyme sites and thereby inhibiting vital metabolic pathways.
  • antimetabolite nucleobase quite broadly to refer to any purine or pyrimidine-based molecule that will effect an anticellular, antiviral, antitumor, antiproliferative or antienzymatic effect, regardless of the underlying mechanism.
  • Exemplary- structures are shown in Table 2, including preferred conjugates such as purine or pyrimidine antimetabolites such as FU, AraC, AZT, ddl, ddC, xylo-GMP, Ara-AMP, PFA or LOMDP, and phosphorylated versions thereof.
  • preferred conjugates such as purine or pyrimidine antimetabolites such as FU, AraC, AZT, ddl, ddC, xylo-GMP, Ara-AMP, PFA or LOMDP, and phosphorylated versions thereof.
  • Preferred compounds for conjugating to a texaphyrin include naturally-occurring purine or pyrimidine nucleobases, namely, cytosine, guanine, thymidine, adenine and uridine. Equally, such texaphyrin conjugates may include modified versions of any of these, such as the heterocyclic components of those nucleoside/ nucleotide analogues listed in Table 2 and phosphorylated version of the compounds listed therein.
  • texaphyrin mononucleobase derivatives including chemically modified nucleobase such as "protected" bases.
  • Protecting groups are used to protect reactive groups, such as amino and carboxyl groups, from inappropriate chemical reactions.
  • Texaphyrin-nucleobase conjugates with protected bases include, for example, conjugates wherein one or more base has a protecting group, such as 9-fluorenylmethylcarbonyl, benzyloxycarbonyl, 4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl, 1-adamantyloxycarbonyl, benzoyl, N-triphenylmethyl or N- di- (4-methoxyphenyl)phenylmethyl on the amino group of the nucleobase.
  • a protecting group such as 9-fluorenylmethylcarbonyl, benzyloxycarbonyl, 4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl, 1-adamantyloxycarbony
  • Conjugation of a nucleobase to a texaphyrin derivative to form a mononucleobase texaphyrin conjugate may be via any of the R groups shown in Structure I. Conjugation of the two separate nucleobases to a texaphyrin derivative to form . a dinucleobase texaphyrin conjugate may also be via any two of these R groups. Any of the texaphyrin mono-, di-, or oligo-nucleobase conjugates and derivatives, including both naturally- occurring nucleobases and modified nucleobases, may also be linked to a solid-support to form a matrix-supported texaphyrin composition.
  • nucleobase conjugates by the condensation of texaphyrin mono- and bis-acids with conveniently modified nucleobases
  • various spacers may be used. These include, for example, oligomethylene bridges with terminal amino, or hydroxy function, which allow formation of an amide or ester bond for the connection of the texaphyrin and nucleobase units. This bridge may also be modified, e.g., by the reduction of the amide bond to give the amine function.
  • the present invention thus encompasses many possibilities for the connection of the same or different nucleobases to texaphyrin macrocyc1es.
  • expanded porphyrin- nucleobase conjugates will have a wide variety of applications, ranging from the use of texaphyrin-derived compounds as agents for selectively delivering an associated, biologically active nucleobase to a particular body, or even subcellular, locale to the more general use in laboratory protocols concerned with nucleotides or oligonucleotides.
  • the texaphyrin-nucleobase conjugates will act to deliver the antimetabolite to subcellular sites through intrinsic biolocalization, or through a covalently coupled site-directed molecule, such as an oligonucleotide, peptide, or hormone, for example.
  • Expanded porphyrin-nucleobase constructs may employ only one nucleobase-containing substituent for each macrocycle; however, this is in no way a limitation upon the invention.
  • texaphyrin-nucleobase conjugates of the present invention may have any number of nucleobases or nucleobase oligomers or polymers attached. The choice of base would be generally governed by the application in which the conjugate was to be primarily used. Thus, it is contemplated that a texaphyrin-nucleobase conjugate with adenine will selectively bind thymidine, presumably through a hydrogen bonding of the two nucleotides.
  • polymers of texaphyrin-nucleobase conjugates can be constructed and used to bind oligo- or poly-nucleotides through complementarity with the sequence of bases "encoded" on the texaphyrin-nucleobase polymer.
  • X is the texaphyrin macrocycle
  • N is the conjugated nucleobase structure
  • Y is the hydrogen bonded poly- or oligonucleotide.
  • texaphyrins of the present invention may serve as a carrier for polymers of nucleobases, wherein the nucleobase polymers are attached covalently to the texaphyrin macrocycle, such as might be exemplified through the structural designation:
  • X is a single texaphyrin macrocycle
  • N is a selected oligomeric or polymeric nucleotide or other nucleobase
  • Y is a hydrogen bonded poly- or oligonucleotide
  • Such structures would be useful in a number of contexts.
  • texaphyrins they are currently envisioned to be useful in RNA hydrolysis.
  • sapphyrins they may be used as specific carriers for taking complementary nucleotides into cells.
  • the complementary nucleotides could be structures such as antisense molecules, including C-5 propyne-containing antisense oligonucleotides, designed to inhibit the transcription, translation or both, of a given gene or construct. Alternatively, they could be coding for
  • sense strands of DNA which encode an entire gene, a functional protein domain, or any polypeptide, peptide, or fragment thereof.
  • Such constructs may be used in in vi tro molecular biological embodiments or in gene-transfer protocols in which the DNA is intended to act as a template for the production of proteins or peptides such as normally-functional or therapeutically- important proteins and peptides.
  • any number of nucleobase structures can be attached to a texaphyrin macrocycle.
  • the ultimate number of such residues that are attached will, of course, depend upon the application.
  • one intends to employ such a structure to purify complementary nucleotides one may well desire to employ a structure having a polymer of at least 10 or so bases attached.
  • the nature of the intended use will dictate the number of attachment points there are on the texaphyrin macrocycle for attaching nucleobase moieties.
  • a single attachment site will suffice for most applications, for certain particular applications those of skill may find it particularly advantageous to attach various nucleobases at various of the subcomponents of the macrocycle.
  • oligonucleotides are currently purified using polyacrylamide gel electrophoresis (PAGE) which is time- consuming, uses toxic materials, and is low-yielding.
  • PAGE polyacrylamide gel electrophoresis
  • Ion exchange columns can, on a limited basis, sometimes separate oligonucleotides containing 40 or fewer residues. However, this process is also generally unsuitable as, for example, severe conditions such as elution at pH 2.7, are often required.
  • the availability of a new column material, or improved functionalized glass capillaries, for use in chromatographically or electrophoretically separating nucleotides and oligonucleotides would thus represent a significant breakthrough in this area.
  • oligonucleotide-derived products are currently being used as probes for the detection of genetic diseases and for proviral HIV, the causative agent of Acquired Immunodeficiency Syndrome (AIDS) 2 . They are also being considered as potential chemotherapeutic agents, both directly, i.e., in gene therapy, and in an antisense fashion. 3
  • silica gel phases with bonded groups such as linear hydrocarbons, amino groups, cyano-groups, carboxylic amides and amino acids are all known. Unfortunately, few if any, of these phases are efficacious for the efficient, high-yielding separation of nucleotides and oligonucleotides.
  • ion exchange columns which, on a limited basis, can sometimes separate oligonucleotides containing 40 or fewer residues.
  • this technique still suffers from several limitations, not least that it requires severe conditions, including elution at pH 2.7, for routine operation 8 .
  • commercially available hydroxyapatite columns are limited for low pressure only (up to 719 psi) .
  • Reverse-phase columns are designed for pressures up to 5000 psi.
  • this separative technique is based on hydrophobic interaction and, because there is not a specific interaction for phosphorylated species, it is not surprising that effective separation is not generally achieved.
  • the matrix-supported expanded porphyrins of the present invention will be bonded, texaphyrin-substituted glass capillaries or silica gels that will serve as specific phosphate- or nitrate-chelating solid-supports. These may be used in the chromatographic and electrophoretic separation of nitrates, phosphates and phosphorylated species, including nucleotides, oligonucleotides, and DNA or RNA molecules including gene fragments, and will be suitable for both analytical and preparative HPLC applications.
  • Adenylate Energy Charge TATPl + 0.5 TADPl (1)
  • a great advantage afforded by these aspects of the invention is that different numbers of texaphyrin molecules may be introduced per polymer unit simply by varying the molar ratio of texaphyrin derivative to the number of the groups bonded on the polymer surface.
  • the inventors therefore, also propose to vary the micromolar concentration of texaphyrin used per gram of silica gel in order to control the retention times and to influence peak shape characteristics. Straightforward modifications such as these will thus allow such systems to be used in both analytical and preparative modes.
  • the inventors envision the separation of oligonucleotides using a matrix-supported metallotexaphyrin having little or no hydrolytic or cleavage activity towards oligoribonucleotides or oligodeoxyribonucleotides.
  • Gadolinium is a preferred metal for this separation, however other metals such as lanthanum, lutetium, neodymium and yttrium are expected to be effective.
  • these new chromatographic supports are suitable for use in analyzing and purifying a large number of critical products, including small molecule antiviral agents and oligonucleotides such as hybridization probes and primers. They represent a marked advance over existing technology including PAGE, HPLC, HPAC, ion exchange columns and purine and pyrimidine base-bonded silica gels, and are expected to find many immediate applications in DNA-related biotechnology and medicine.
  • Such matrix-supported texaphyrins are also contemplated for use in separating very large RNA or DNA molecules such as gene fragments and antisense constructs.
  • various elution-related parameters such as, e.g., flow rate, buffer strength, and texaphyrin-to-silica loading level, may be optimized so as to achieve the best possible separations.
  • Second and/or third generation polymer-supported expanded porphyrins may also be prepared for use in connection with RNA or DNA separation and purification.
  • the second generation stage of this new synthetic development may be considered to be the stage of long chain alkyl addition.
  • Long chain alkyl groups will thus be introduced onto the surface of the texaphyrin- substituted silica-gel. This will result in columns that will combine the properties of both reverse phase separation and texaphyrin-based phosphate chelation and ion exchange.
  • the inventors contemplate using appropriate synthetic modifications to optimize the retention characteristics of the columns so that they separate selectively any desired length of oligonucleotide and/or gene fragment.
  • the yet superior third-generation systems contemplated by the inventors are those in which the stationary phases will combine the best features of three different known modes of separation: hydrogen bonding, electrostatics, and reverse phase hydrophobicity. This will be achieved by using various different substituents on both the texaphyrin molecules and on the stationary phase material itself.
  • these groups may be varied so as to introduce a nucleobase or nucleobase analog.
  • nucleobase-bearing systems are expected to act in conjunction with the combined texaphyrin electrostatic and reverse phase hydrophobic interactions described above.
  • the resultant solid phase will be able to separate nucleotides and oligonucleotides not only on the basis of charge and length but also on the basis of nucleic acid type.
  • Such phases would prove to be of tremendous value in applications involving the analysis and purification of gene fragments since these are often of similar size but of very different chemical (i.e., nucleic acid) composition.
  • the inventors also contemplate using polymer-supported texaphyrins in connection with capillary electrophoresis (CE) .
  • CE capillary electrophoresis
  • the polymer will be an untreated glass capillary rather than silica gel. Since glass is chemically similar to silica gel (both are silicate-derived) , the preparation of a range of modified glass surfaces can be straightforwardly achieved in light of the present disclosure.
  • Such texaphyrin-glass constructs may be used to separate natural and synthetic nucleotides and oligonucleotides.
  • the best combination of complexed metals and substituent groups needed to effect any given type of separation will be determined.
  • the information gained using silica gels will be applied in these considerations and supplemented by further straightforward studies. It is expected that this will lead to a significantly improved new CE system.
  • the inventors contemplate carrying out a Sanger sequencing of pBR322 using fluorescently labeled dideoxy nucleotides (Applied Biosystems Inc.). Since an entire range of fragment sizes is produced here, this will allow the superiority of the new texaphyrin-based approach to be quickly demonstrated.
  • texaphyrin-based supports As described above and in the detailed examples, the inventors designed and constructed texaphyrin-based supports and used them to achieve separation of nucleotides, thus overcoming many of the existing drawbacks described above. Similar methods using a polymer-supported texaphyrin are envisioned to be of use purifying plasmid DNA, as described in Example 7D.
  • oligonucleotide analysis Another technical area in which significant improvements could be made is in oligonucleotide analysis.
  • automated gene sequencing is currently carried out using either radio- or fluorescent- labeled nucleic acid gel electrophoresis. This technique is limited by the requirement for either slab or tubular polyacrylamide gels.
  • An alternative approach, currently being considered on a research basis, is to use capillary electrophoresis. Unfortunately, this is limited when it comes to achieving separations based purely on electrostatics and oligomer size.
  • Texaphyrins linked to glass capillary columns, as disclosed herein, provide new devices and methods that allow for such separation.
  • a further utility of polymer-supported expanded porphyrins i.e., texaphyrins and sapphyrins is their use as tools in the removal of phosphate- or nitrate- containing environmental contaminants from ground water, soil, foodstuffs, and the like.
  • the sapphyrins and texaphyrins have different binding mechanisms that may render one more useful in certain embodiments. For example, one may prefer to use a sapphyrin polymer- support to remove phosphates or nitrates from solutions.
  • Metallotexaphyrins conjugated to solid-supports offer a unique and distinct way of effecting the recognition and purification of anionic and neutral substrates.
  • both texaphyrin and sapphyrin columns will likely bind and remove a variety of anionic compounds, with texaphyrins also binding to and being able to purify neutral molecules, such as sugars, including sugars present within the blood stream.
  • Texaphyrin and sapphyrin matrix-supports may be employed to analyze and separate pesticides such as Dichlorovos, Phosphamidon, Diazanon, and Parathion, herbicides and fungicides, many of which contain organophosphorus compounds.
  • Texaphyrin-substituted gels and columns may even be employed in the rapid detection and analysis of organophosphorus chemical warfare agents, allowing them to be disposed of where necessary.
  • Anionic species such as nitrates, phosphates and various metals elements are major sources of environmental pollution.
  • an excess of nitrate can cause an imbalance of the water chemistry and lead to the formation of excess algae which can threaten the balance of the entire ecosystem.
  • excess nitrates in soil can lead to poor growth conditions for crops. Therefore, a system to remove excess nitrates, phosphates and metals from environmental sources would be of great benefit for retaining the delicate balance in these ecosystems.
  • the ability to produce a wide range of matrices is dependent on the ability to synthesize active catalysts. These catalysts must be able to convert the appropriate monomers, such as ethylene or styrene, to the desired polymeric materials.
  • Lanthanide texaphyrin complexes have potential as active polymerization catalysts. There are several reasons for this. First, the texaphyrin ligand is essentially planar with the metal being slightly above the plane defined by the five chelating nitrogens, a geometry that allows access to the metal from either side of the macrocycle. Second, because the total coordination number of a metallotexaphyrin complex is high while that of the ligand itself is low, the texaphyrin complexes should allow substrates such as alkenes or dihydrogen to be easily accommodated. Finally, the catalysts proposed herein will have two potential active sites.
  • the present invention now allows the lanthanide texaphyrin (txph) complexes, as linked to solid-supports, to be used as the basis for preparing a new set of potential polymerization and hydrogenation catalysts. These compounds may now, for the first time, be employed in heterogeneous catalysis, as described below.
  • the texaphyrins when supported on solid matrices, provide a source of unparalleled Lewis acidity as a result, primarily, of the fact that trivalent cations of the lanthanide series are far better Lewis acids than cations of the more common metals.
  • This Lewis acidity allows for the use of texaphyrins, either free in solution or supported on supports, in a range of catalytic applications. These range from such standard catalytic applications of Lewis acidity as acyl transfer, ester hydrolysis, and amide hydrolysis to more sophisticated embodiments as phosphomono- and phosphodiester hydrolysis.
  • the critical catalytic step is thought to involve, in a mechanistic sense, the binding of a carbonyl or heteroatom-containing carbonyl- like oxygen lone pair to the coordinated texaphyrin metal center.
  • This binding in accord with the widely accepted principles of Lewis acid catalysis, will serve to activate the already acidic carbonyl carbon or heteroatom (e.g., phosphorus) for subsequent attack by a nucleophile.
  • nucleophile is provided by water, hydrolysis ensues; when it is provided by an alcohol, amine, or other standard nucleophilic organic group, acyl transfer results.
  • these catalytic systems as normal for all catalysts (principle of microscopic reversibility) can be used to both make and break bonds.
  • the stability of the matrix-supported texaphyrin complexes allows them to be subjected to harsh conditions, such as organic solvents and elevated temperatures, without breakdown of their structure.
  • This property imparts a particular usefulness to the range of polymer-supported metallotexaphyrins and, for example, allows them to be used as catalysts in various embodiments where the use of a biological catalyst, such as an enzyme, is impossible due to their instability and/or temperature sensitivity.
  • Samarium (III) complexes are envisioned to be particularly useful due to the relative ease of characterization compared to the remaining lanthanide complexes.
  • samarium complexes typically have narrow line widths in NMR spectra even though the metal center is paramagnetic. Therefore 1 H and 13 C NMR spectra may be used as standard characterization tools. In fact, this is the case for Sm(txph) (N0 3 ) 2 which exhibits an 1 H NMR spectra resembling that of a diamagnetic complex.
  • the Gd(III) complexes with their known high relaxivity- inducing abilities will be useful for MRI applications.
  • Gd(III) and Sm(III) and other complexes other characterization techniques will include IR, UV/vis, mass spectroscopy, elemental analysis and, when appropriate, single crystal X-ray diffraction analysis. Polymeric or oligomeric materials obtained from these catalysts will be examined via standard polymer analyses including mass spectroscopy, GC/MS, gel permeation chromatography as well as those techniques listed above.
  • these new paramagnetic materials When attached to a solid-support, particularly one of a biocompatible nature, these new paramagnetic materials will be of unique use as MRI-detectible catheters. These, in turn, will be of use for medical diagnosis applications. These, or similar, solid-supported materials will also be of use as heterogeneous Lewis acidic catalysts with the usual advantages of site isolation, product purity, and catalyst recovery that would accrue from such heterogeneous catalysts.
  • Non-invasive techniques for the diagnosis of human disease are extremely valuable tools in the practice of modern medicine.
  • One of the most important new techniques available uses magnetic resonance (MRI) to image tissues and organs within the body. This provides an advantage over the use of X-rays, which have harmful side-effects.
  • MRI magnetic resonance
  • a complication of this technique arises when the patient has a catheter, implant or other internal device which needs to be located within the body. At the present time, the majority of these devices are composed of materials that do not produce an adequate signal when subject to MRI. Therefore, catheters and other devices composed of a material which is highly detectible during MRI scans would be of great medical utility.
  • Gadolinium(III) complexes derived from strongly binding anionic ligands, such as DTPA and DOTA, are being developed for use in MRI. Indeed, [Gd»DTPA] " is undergoing clinical trials in the United States for possible use in enhanced tumor detection protocols. Nonetheless, the development of other gadolinium(III) complexes with greater kinetic stability, superior relativity, and/or better biodistribution properties was still a desirable goal until recent times. The provision of water-soluble texaphyrins for use in MRI enhancement, as described in U.S. Patent 5,256,399, incorporated herein by reference, addressed this particular need.
  • AIDS Acquired immunodeficiency syndrome
  • cancer is among the most serious public health problems facing our nation today. AIDS is a fatal human disease that has now reached pandemic proportions. Cancer, in spite of some very significant advances in diagnostics and treatment in recent years, remains the third leading cause of death in this country. Finding better ways to detect, treat, and reduce the transmission of these disorders are thus research objectives of the highest importance.
  • PDT photodynamic therapy
  • Singlet oxygen is also believed to be the critical toxic species operative in experimental photosensitized blood purification procedures.
  • This very new application of photodynamic therapy is of tremendous potential importance: It shows promise of providing a safe and effective means of removing enveloped viruses such as HIV-1, herpes simplex (HSV) , cytomegalovirus (CMV) , various forms of hepatitis-inducing virus, as well as other opportunistic blood-borne infections (e.g. bacteria and malaria plasmodium) from transfused whole blood.
  • AIDS infections do still occur as a result of blood transfusions.
  • banked blood components are essential products for the practice of modern medicine and as a result this method of transmission is not likely to be precluded by simple changes in lifestyle.
  • a means to ensure that all stored blood samples are free of the AIDS virus (and ideally all other blood-borne pathogens) should be developed. To a certain extent, this can be accomplished by screening the donors' histories and carrying out serologic tests.
  • the serologic tests for HIV-1 are insufficient to detect all infected blood samples, in particular, those derived from donors who have contracted the disease but not yet produced detectable antibodies.
  • new mutants of the AIDS virus have been detected; some or all of which may escape detection by current means.
  • an antiviral system which removes any form of HIV-1 from stored blood. This is particularly important since a stored blood sample from one infected donor could potentially end up being administered to several different patients, in, for instance, the course of pediatric care.
  • Texaphyrins are effective as photosensitizers for the generation of singlet oxygen.
  • metallotexaphyrins can be used for the inactivation or destruction of tumors as well as for the prophylactic treatment and removal of human immunodeficiency virus (HIV-1) , and other viral contaminants from blood.
  • HV-1 human immunodeficiency virus
  • the metallotexaphyrins with Cd (II), Sm(III), La(III), Lu(III) have been studied, and the Cd(II) was found to be effective against certain types of leukemia cells.
  • the present invention forms a basis for a novel treatment of diseases, such as leukemia, where the fluid from the body can be removed from the patient and exposed to PDT by use of a column which contains a polymeric supported texaphyrin.
  • the treated fluid can then be reintroduced to the patient without fear of contamination from the texaphyrin. This avoids any unnecessary exposure of the patient's whole system to the effects of the photodynamic therapy.
  • the matrix-supported texaphyrins of the present invention also allow for the deactivation of retroviruses and enveloped viruses in biological fluids, such as blood and blood products, using columns, filters or such like. These methods are advantageous as the fluids may be passed over the column. This may result in improved kinetics, but the very evident advantage is, again, that the treated fluid can be readily recovered and introduced to the patient without fear of contamination from the metallotexaphyrin.
  • sapphyrins with ethylene units could be used, as prepared by the ⁇ elimination of acetoxy derivative, as well as sapphyrin bis acid, sapphyrin diamino and dihydroxyderivatives.
  • Sapphyrins bearing covalently attached nucleobases could also be used for the polymerization reactions.
  • Example 10 of co-pending application, PCT publication WO 94/09003, is specifically incorporated herein by reference to supplement the present example.
  • Radical polymerization may be catalyzed by dibenzoylperoxide, or bisazaisobutyronitril in inert solvent at temperature 120-200°C.
  • Polymeric sapphyrin was obtained also with using sulfolan, hexamethylphosphortriamide as a solvent. The reaction could be also carried out without solvent. The same procedure was used for the sapphyrin dialcohol as a starting compound for the reaction ' with sapphyrin diacid.
  • the new chromatographic bonded phases that are the subject of these aspects of the present invention were prepared in two steps.
  • a phosphate thioether recognition unit was connected (via amide, ether, thioether, or NH bonds) with silica gel.
  • the density of coverage for the covalently bonded recognition groups on the silica gel was controlled by the amount of expanded porphyrin, or guanidinium derivatives (in mmol) that was attached to 1 g of the starting silica gel.
  • Elemental analysis data namely comparison of the %C and %N in the starting material and the product, provided a measure of the ratio of phosphate receptor (in ⁇ M) to 1 g of silica gel.
  • Solid state 31 P NMR spectra also provided a unique tool for the detailed study of the mechanism of phosphate binding.
  • Example 11 of co-pending application PCT publication WO 94/09003, is specifically incorporated herein by reference to supplement the present example.
  • silica gel For the separation of highly polar phosphorylated species on both analytical and preparative scale, the surface of silica gel must be modified by a silylation procedure. This procedure basically converts most, if not all, the free silanol groups (-Si-OH) to -Si-O-Z type residues, where Z is an alkyl chain, or aryl substitution (ArC0 2 -, ArCO-) .
  • Silylation reagents which have been employed are those of type Z 2 SiCl 2 , ZSiCl 3 , and Z(CH 3 ) 2 SiCl, where Z is an alkyl residue with 1 to 18 carbon atoms in it or an aryl (phenyl, pentafluorophenyl) substituent.
  • the present inventors have used the following procedure: Introduction of the C 1# C 8 , C 12 , and C 18 groups by reaction of mono and trichlorosilanes with suspended bonded silica gel in an organic solvent (dry dichloromethane, dichloroethane, benzene, toluene, xylene, or pyridine and 2,6-lutidine was used directly as a solvent) either directly or in the presence of organic bases such as pyridine or 2, 6-lutidine, in a temperature range of 25°C-150°C for the preparative HPLC phases, the corresponding triflates were used for silylation.
  • an organic solvent dry dichloromethane, dichloroethane, benzene, toluene, xylene, or pyridine and 2,6-lutidine was used directly as a solvent
  • organic bases such as pyridine or 2, 6-lutidine
  • Example 12 of co-pending application PCT publication WO 94/09003, is specifically incorporated herein by reference to supplement the present example.
  • texaphyrin-bonded silica gel was accomplished by amide bond formation between activated (Eu) texaphyrin carboxylic acid (carbodiimide method) and amino-substituted silica gel.
  • the sp 3 texaphyrin derivative T2B1.HC1 (having 0(CH 2 ) 3 C0 2 H as an R substituent on the B portion of the molecule, 0.694 g, 1 mmol, see U.S. Patent 5,252,720 for T2B1 structure) was dissolved in 80 ml of dry methanol .
  • Eu (OAc) 3 .H 2 0 (0.329 g, 1 mmol) was added, followed by triethylamine (0.5 ml).
  • the reaction mixture was heated at reflux with the reflux condenser open to the air for 6 hours, with the progress of metallation being followed by visible spectroscopy.
  • the methanol solvent was evaporated off under reduced pressure to give a dry, dark solid that was washed with dichloromethane for 2 hours under conditions of vigorous stirring.
  • the product was filtered off, redissolved in MeOH (25 ml) , and the solution was treated with zeolite (a procedure for removing free europium salt) .
  • the product 4a was then twice precipitated from methanol by adding diethylether. The resulting dark green solid was then collected and dried under high vacuum overnight. The yield was 91.0%.
  • the reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 3 days.
  • the product was filtered off, washed with dichloromethane (30 ml) , methanol (50 ml) , water (100 ml) , methanol (50 ml) , and dichloromethane (30 ml) .
  • the product 4b was dried (giving 1.035 g) and used for further derivatization, namely direct silylation, or 1) introduction of other functional groups and then 2) silylation.
  • M - trivalent metals such as YOU), In(III), Ln( ⁇ i), where Ln - Lanthanide; H2 N-R represents an arnmo-functionalized solid support
  • the preparation of improved second and third generation texaphyrin solid-supports may be achieved in many ways.
  • long chain alkyls may be introduced onto the surface of the texaphyrin-substituted silica-gel. This is contemplated to result in second-generation columns that will combine the properties of both reverse phase separation and ion exchange.
  • third generation texaphyrin solid-supports may also be prepared.
  • third-generation systems may be prepared in which various different substituents on both the expanded porphyrin molecules and on the stationary phase material itself are chosen to impart the desired groups and properties to the resultant material.
  • nucleobase-bearing systems may be generated which are expected to be able to separate nucleotides and oligonucleotides not only on the basis of charge and length but also on the basis of nucleic acid type.
  • the use of nucleobase-bearing systems to hydrolyse RNA oligonucleotides with sequence specificity is also contemplated.
  • the introduction of aryl substituents give the possibility for improved separation based on phosphate chelation and ⁇ - ⁇ stacking.
  • Any substituent for multiple type separation may be introduced by acylation, akylmethylation on free amino groups and/or by silylation with phenyl (substituted phenyl) , silyl reagents or arylmethylation of OH groups.
  • an imidazole ring can be easily introduced by reaction with aminoprotected (e.g. 1-BOC-L-Histidine) .
  • a positive charge near the texaphyrin macrocycle can also be introduced by reaction with, for example, L-arginine methylester.
  • Example 14 of co-pending application, PCT publication WO 94/09003, is specifically incorporated herein by reference for the purposes of supplementing the present example in terms of the separation of negatively-charged species using expanded porphyrin matrix-supports.
  • Texaphyrin columns are prepared using a standard column packing procedure. In this context, the columns are slurry-packed in methanol or acetone, followed by a wash with water. An HPLC pump that generates a solvent flow of about 10 ml/min at 300-400 bar is used.
  • the inventors were able to successfully separate adenosine and its mono-, di-, and tri-phosphorylated nucleotide forms.
  • An adenosine, AMP, ADP, and ATP mixture, (20 ⁇ l) was loaded onto and eluted off the sapphyrin-modified silica gel using an isocratic buffer of 100 mM dibasic ammonium phosphate buffer at a pH of 7.0 and a flow rate of 0.2 ml/min (or, in another study, 500 mM buffer and a flow rate of 1.0 ml/min) .
  • the wavelength of 260 nm was monitored and results were confirmed with retention times and UV-visible spectrum obtained from samples of individual compounds.
  • silica- bound lanthanide texaphyrins specifically, europium(III) texaphyrin can effectively separate mono-, di-, and polyanionic species under isocratic HPLC conditions.
  • the monoanionic cyclic AMP eluted at nearly the same time as the neutral adenosine.
  • the other phosphate derivatives showed a much higher affinity for the solid phase than the monoanionic cyclic AMP.
  • the present data show that the expanded porphyrin, texaphyrin, when metallated with a lanthanide metal, was able to effectively separate adenosine, AMP, ADP and ATP. Since crystal structures of dysprosium texaphyrin show that phosphate anions ligate to the dysprosium via axial binding, the inventors believe that the separation is via axial ligand binding to the lanthanide metal in the texaphyrin macrocycle. Varying the metal in the macrocycle is expected to achieve varying degrees of selectivity between anions.
  • the silica-bound texaphyrin hydrolyzed the phosphodiester bond between the nucleotides. Varying the metals in the solid-support bound texaphyrin macrocycle will vary the rate of hydrolysis and the specificity of hydrolysis.
  • the inventors were able to successfully separate 2-, 3-, 4-, 5-, 6-, 7-, 8- and 9-mer oligonucleotides.
  • nitrates The binding of nitrates is contemplated to be of significance in removing nitrates from water samples, including, for example, drinking water, particularly for babies, and also water in fish tanks and aquariums - there is currently no way of achieving this.
  • removal of both nitrates and phosphates, such as alkylphosphonates and detergents, from contaminants of waste water or ground water is also contemplated.
  • Metallotexaphyrins conjugated to solid-supports offer a unique new way of effecting the recognition and purification of anionic substrates. This is because they possess, coordinated within their central cores, large metal cations that necessarily (by definition) are Lewis acidic and hence able to bind, as apical or axial ligands various Lewis basic entities, including those that are negatively charged.
  • the degree of this Lewis acid-to-Lewis base interaction is a function not only of the particular metal center coordinated to the central, pentaazatexaphyrin core, but also of the Lewis base itself, since the latter could vary in the degree of basicity or ligand donor strength as well as in size and steric accessibility.
  • individual Lewis basic materials will be retained differentially.
  • Texaphyrins linked to solid-supports therefore provide highly selective means for separating species from solutions of different anionic materials.
  • a solution is contacted with the texaphyrin-based solid- support and subjected to standard elution procedures, distinct components will be released at different rates. They can then be collected as small eluent fractions to allow for the obtainment of the various components of the original anion mixture in a partially or completely purified fashion.
  • Metallotexaphyrins as is apparent from inspection of numerous crystal structures 1,13 , also bind neutral entities as apical ligands provided that these entities are electron rich (i.e., Lewis basic) .
  • Lewis basic electron rich
  • texaphyrins conjugated to solid-supports can also be used to effect the chromatographic or electrophoretic separation of neutral, Lewis basic entities.
  • different neutral substrates differ in their ligation ability (because of differences in Lewis basicity, sterics, etc.) that makes such separations possible.
  • oligonucleotide and other polyphosphorylated substrates represents a specific embodiment of the above Lewis base-to-texaphyrin based procedure.
  • the major advantage that the presently disclosed approach would provide over the currently available methods is an ability to separate ostensibly similar materials on the basis of total phosphate number.
  • oligonucleotides, for instance, of different length would present to the texaphyrin-bearing surface a number of total interactive axial ligands that differ absolutely.
  • the degree of interaction between the texaphyrin surface and the substrate in question i.e., an oligonucleotide of different length.
  • the order of elution would be different in a chromatographic or electrophoretic sense and a means for differential purification established.
  • lanthanide (III) texaphyrin (LnTx) functionalized polymers involves their utility in the separation of plasmid DNA from RNA, protein, and other cellular contaminants.
  • Current protocols describe procedures for lysis of bacterial cells and provide a source of crude plasmid DNA (e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning, 2nd
  • RNA-supported texaphyrins presents a novel alternative method of purification, whereby unwanted RNA contaminants are removed by virtue of their susceptibility to hydrolysis by the lanthanide complexes.
  • Treatment with the solid-supported LnTx catalyst therefore results in the degradation of RNA to mononucleotides, which are readily removed using a standard ethanol precipitation.
  • Unwanted protein is also readily removed by virtue of its relatively low affinity for the solid-supported LnTx.
  • the crude nucleic acid pellet obtained by the boiling, SDS, or alkaline lysis methods (Sambrook, 1.34- 1.39), is resuspended in 3 mL of Tris EDTA buffer, pH 8.0.
  • Tris EDTA buffer pH 8.0.
  • An appropriate amount of, eg., silica bead supported LnTx is added to this solution, the suspension vortexed, and incubated in a heat block at greater than 37°C for a period of time sufficient to effect complete hydrolysis of the RNA.
  • the suspension is then cooled, washed into an eppendorf pipette tip to form a small column using Tris-EDTA buffer, and washed briefly to remove remaining protein.
  • the purified DNA is then eluted from the solid-supported LnTx using a sodium phosphate buffer.
  • the resulting DNA is precipitated using one-tenth volume of 3 M sodium acetate, pH 5.4 and three volumes of ethanol.
  • the supernatant is decanted, the DNA pellet washed with 70% ethanol, and allowed to air-dry.
  • the purified DNA is resuspended in any desired buffer.
  • the present example sets forth various ways in which matrix-supported metallotexaphyrins may be employed to catalyze a variety of chemical transformations.
  • Solid-supported lanthanide texaphyrins provide a unique means of catalyzing the hydrolysis of phosphodiesters.
  • the Lewis-acidic lanthanide metals coordinated within the texaphyrin bind the phosphate, thus activating the phosphate towards hydrolytic cleavage.
  • a suspension of the solid-supported lanthanide texaphyrin can be treated with a solution of a phosphodiester (such as RNA) resulting in the hydrolysis of the phosphodiester.
  • the catalysts may then be easily removed by a simple filtration. Thus, products may be easily separated from the catalysts.
  • Example 4 The fact that europium texaphyrin attached to a solid-support can catalytically hydrolyze phosphodiester bonds at neutral pH was demonstrated in Example 4. Therefore, two-phase catalytic hydrolysis of phosphodiesters is possible.
  • a stirred suspension of solid-supported lanthanide texaphyrin alkyl complexes may be treated with an appropriate monomer solution (ethylene, styrene, acetylene, phenylacetylene, etc.) resulting in polymer formation. Additional monomers (different from the initial monomer) may be added to this solution resulting in a block polymer. After complete polymerization has occurred the catalyst may be removed by filtration resulting in a purified polymer suitable for a variety of applications including household plastics or machine parts.
  • an appropriate monomer solution ethylene, styrene, acetylene, phenylacetylene, etc.
  • a hydrogen-saturated solution of alkene will be stirred with a suspension of the solid-supported lanthanide texaphyrin hydride complexes under a hydrogen atmosphere.
  • the alkene will be hydrogenated to the corresponding alkane.
  • the catalyst may then be removed by a simple filtration step resulting in a purified alkane suitable as a feed stock for the petroleum and chemical industries.
  • EXAMPLE 9 MATRIX-SUPPORTED TEXAPHYRINS FOR RACEMATE RESOLUTION
  • the present example describes the use of matrix-supported texaphyrins as chiral sorbents for HPLC.
  • a sorbent for racemate resolution can be generated.
  • Specific examples of chiral groups are D- glucopyranosyl, glucosamine, L-amino acids, such as L- alanine, or L-phenylalanine residues, oligopeptides, oligosaccharides and chiral binaphthyl systems.
  • the resultant chiral matrix-supported texaphyrins could be used for D,L-aminoacid separation, as well as for the separation of chiral phosphates, such as synthetic nucleotide monophosphates with modified ribose units, phospholipids, phosphoproteins, phosphorylated saccharides, and the like.
  • chiral phosphates such as synthetic nucleotide monophosphates with modified ribose units, phospholipids, phosphoproteins, phosphorylated saccharides, and the like.
  • These same polymer-supported texaphyrin chiral sorbents may be also used to effect catalytically a range of enantiotopic reactions, from chiral phosphate ester hydrolysis to stereoselective polymer genesis.
  • Texaphyrin acid (1 mole, eq.) was dissolved in dry DMF and the solution cooled to 0°C for 1 hour. The reaction mixture was then stirred at room temperature for 2-5 days, with the course of the reaction being followed by HPLC. The product was isolated by filtration and washing.
  • This example sets forth various ways in which matrix-supported metallotexaphyrins may be used in medical devices, such as catheters, to enhance visibility during magnetic resonance imaging.
  • nonlabile Gd(III) complexes of hydroxy-substituted texaphyrins are useful contrast agents for MRI applications.
  • MRI can provide two-dimensional sectional images through a patient, providing color or gray scale contrast images of soft tissue, particularly for imaging tumors, edema, infarcts, infections, and the like.
  • magnetic resonance images are particularly desirable since they do not expose the patient to harmful radiation.
  • catheters, tubes, implants, and other devices present within their bodies, and the precise anatomical locations of such devices can be of substantial clinical importance.
  • most catheters and many other devices are composed of materials, such as organic polymers, which do not produce adequate signals for detection by magnetic resonance imaging techniques.
  • most polymeric catheters are not clearly discernible on magnetic resonance images unless they are surrounded by tissue that has a high signal intensity, in which case they leave a dark void on the image. It is therefore highly desirable to provide catheters and other medical devices that have enhanced detectability when viewed using MRI regardless of the nature of the surrounding tissue.
  • a texaphyrin complexed to an imaging ligand, such as Gd(III), and then linked to a catheter or device would be of great use in providing a high contrast image when viewed under MRI.
  • Texaphyrins may be linked to catheters and devices by the same basic synthetic methodology that is employed, and described herein, to link texaphyrins to any other polymeric matrix.
  • the ions used for MRI may be any metal ion that displays paramagnetic properties and that binds to a texaphyrin, with exemplary transition metal cations including Gd(III) , Cu(II), Ni(II), Co(III), Co(II), Fe(III), and Fe(II), with Gd(III) being preferred.
  • the polymeric materials particularly suitable for use in such catheters and devices include, for example, polyethylene, polyurethane, polyvinyl chloride, nylon, latex, silicone rubber, halogenated polyethylenes (e.g., polytetrafluoroethylene (PTFE) and other Teflon ® materials), organosilicones (e.g., Silastic ® materials) and biocompatible ceramics.
  • halogenated polyethylenes e.g., polytetrafluoroethylene (PTFE) and other Teflon ® materials
  • organosilicones e.g., Silastic ® materials
  • the combination After linking the metallotexaphyrin to the polymeric material, the combination would then be formed into a tube, or shaped device, by conventional techniques, such as, e.g., injection molding or extrusion at elevated temperatures.
  • Suitable extruders utilize polymeric materials and, by applying heat and pressure, form the materials to a continuous length of tubing having a desired diameter and wall thickness.
  • the metallotexaphyrins of this invention may be incorporated throughout such tubes uniformly, or may be located only in a portion of the tube, such as a distal portion, or in a plurality of circumferential bands, e.g., axially spaced apart along the tube.
  • paramagnetic ions can be provided along an axial line or strip of the flexible tubing, e.g. by introducing the texaphyrins into the extruder at one circumferential region of the tube as it is extruded.
  • Texaphyrins may also be used to modify portions or components of permanently implantable devices, such as joint and other prostheses, breast implants, pacemakers, drug injection ports, pediatric intercardiac devices and even drug delivery devices, where it is desirable that the presence and location of the device be readily discernible during subsequent magnetic imaging procedures.
  • the more stable, polymer-supported texaphyrins of the present invention may be employed in radiosensitization and therapy (RIT) , where the radiometal of interest must be bound and retained under physiological conditions.
  • RIT radiosensitization and therapy
  • the potential damage arising from "free" radioisotopes, released from the complex, can be very serious.
  • the advantage of a chelate, and particularly, a polymer-supported texaphyrin metal complex, that does not allow for metal release is clear.
  • an ideal isotope should be readily detectable by available monitoring techniques and induce a minimal radiation-based toxic response.
  • these and other necessary requirements implicate the use of a ⁇ -ray emitter in the 100 to 250 KeV range, that possesses a short effective half-life (biological and/or nuclear) , decays to stable products, and, of course, is readily available under clinical conditions.
  • Texaphyrin forms a kinetically and hydrolytically stable complex with In(III) ; such a ligand system may be elaborated and serve as the critical core of a matrix- supported texaphyrin conjugate for use in 11:1 In-based radioimmunodiagnostics.
  • a matrix- supported texaphyrin e.g., a highly directable, small catheter, could be employed to deliver a radiotherapeutic insult to the center of a solid tumor that is not readily targeted by agents present in the blood.
  • Texaphyrins having electron donating groups on the 2, 7, 12, 15, 18 and/or 21 positions of the present invention are particularly suited for this application due to their enhanced stability.
  • a texaphyrin complexed to 90 Y may be administered in combination with another texaphyrin complexed to a diamagnetic metal for photodynamic tumor therapy, for example, to achieve a synergistic killing of malignant cells.
  • This example sets forth the uses of matrix-supported texaphyrins in photodynamic therapy (PDT) , as may be used, for example, to kill certain types of leukemia cells and to inactivate viruses.
  • PDT photodynamic therapy
  • One example of the utility of the present invention is the use of matrix-supported texaphyrins for photon- induced deactivation of viruses and virally-infected or potentially- infected eucaryotic cells.
  • U.S. Patent 5,252,720 incorporated herein by reference, teaches that unsupported texaphyrins may be used to inactivate peripheral mononuclear cells and enveloped viruses.
  • the columns of this invention are contemplated for use in inactivating viruses such as Herpes Simplex Virus Type 1 (HSV-1) , HIV-1, HIV-2, FIV, SIV, and the like.
  • Texaphyrins having electron donating substituents in the 2, 7, 12, 15, 18 and/or 21 positions of the macrocycle are expected to be more effective photosensitizers for the destruction of free enveloped viruses such as HIV-1, virally-infected peripheral mononuclear cells, as well as leukemia and lymphoma cells.
  • Texaphyrins are photosensitive and unmetallated texaphyrins as well as texaphyrin diamagnetic metal complexes are effective for PDT applications.
  • novel treatment methods made possible by the matrix-supported texaphyrin technology include those in which fluid from the body is removed from the patient and exposed to PDT by use of a column which contains a matrix-supported texaphyrin.
  • the treated fluid may be re-introduced into the patient.
  • the matrix-supported texaphyrins may also be used to remove functional viruses, such as HSV and HIV, from biological fluids, such as blood and plasma, or used as an additional control measure to ensure that blood samples do not contain viruses before storage or transfusions.
  • functional viruses such as HSV and HIV
  • Improved, novel treatment methods made possible by the present matrix-supported texaphyrin invention include those in which fluid from a cancer patient is removed and exposed to PDT using a column that contains a polymer-supported texaphyrin. The treated fluid will then be re- introduced into the patient. This is particularly suitable for purging tumor cells from the blood of patients with leukemia.
  • Matrix-supported expanded porphyrins may be prepared in which the matrix is a glass capillary, for example, as may be used in capillary electrophoresis (CE) .
  • the preparation of modified glass surfaces may be achieved generally as described herein.
  • the preparation of texaphyrin modified 3-amidosubstituted glass and texaphyrin modified arylamine glass would be essentially as described in Example 17 of co-pending application, PCT WO 94/09003, that is specifically incorporated herein by reference for the purposes of supplementing the present example in terms of the preparation of second and third generation matrix- supported texaphyrins.
  • Amides, ethers and thioethers are representative of linkages that may be used for coupling oligonucleotides to texaphyrin metal complexes.
  • Oligonucleotides functionalized with amines at the 5' -end, the 3'-end, or internally at sugar or base residues are modified post- synthetically with an activated carboxylic ester derivative of the texaphyrin complex.
  • oligonucleotide analogues containing one or more thiophosphate or thiol groups are selectively alkylated at the sulfur atom(s) with an alkyl halide derivative of the texaphyrin complex.
  • 01igodeoxynucleotide-complex conjugates are designed so as to provide optimal catalytic interaction between the RNA or DNA phosphoester backbone and the matrix-supported texaphyrin-bound lanthanide cation(s) .
  • Oligonucleotides are used to bind selectively compounds which include the complementary nucleotide or oligo or poly-nucleotides containing substantially complementary sequences.
  • a substantially complementary sequence is one in which the nucleotides generally base-pair with the complementary nucleotide and in which there are very few base pair mismatches.
  • the oligonucleotide will generally be large enough to bind probably at least 9 nucleotides of complementary nucleic acid.
  • a general method for preparing oligonucleotides of various lengths and sequences is described by Caracciolo et al . (1989) 12 .
  • the phosphonate based synthesis is conducted by the reaction of a suitably protected nucleotide containing a phosphonate moiety at a position to be coupled with a solid phase-derivatized nucleotide chain having a free hydroxyl group, in the presence of a suitable activator to obtain a phosphonate ester linkage, which is stable to acid.
  • a suitable activator to obtain a phosphonate ester linkage, which is stable to acid.
  • the oxidation to the phosphate or thiophosphate can be conducted at any point during synthesis of the oligonucleotide or after synthesis of the oligonucleotide is complete.
  • phosphonates can also be converted to phosphoramidate derivatives by reaction with a primary or secondary amine in the presence of carbon tetrachloride.
  • the incoming nucleotide is regarded as having an "activated" phosphite/phosphate group.
  • oligonucleotides may also be synthesized using solution phase methods such as triester synthesis. The methods are workable, but in general, less efficient for oligonucleotides of any substantial length.
  • Preferred oligonucleotides resistant to in vivo hydrolysis may contain a phosphorothioate substitution at each base.
  • Oligodeoxynucleotides or their phosphorothioate analogues may be synthesized using an Applied Biosystem 380B DNA synthesizer (Applied Biosystems, Inc., Foster City, CA) .
  • Applied Biosystem 380B DNA synthesizer Applied Biosystems, Inc., Foster City, CA
  • This section provides an example of the utility of the present invention in the hydrolysis of monoesters, in particular, the hydrolysis of UpU, cUMP, 3' -UMP and 2'-UMP. Described are results from studies using the lanthanide (III) T2B2 texaphyrin (U.S. Patent 5,252,720, incorporated by reference herein) . Using the synthetic methodology of the present invention, a matrix-supported lanthanide (III) T2B2 texaphyrin would be constructed and used as described hereinbelow for the unsupported complex. It is contemplated that the matrix-supported version would function essentially as the free version, but would provide the many additional advantages associated with being attached to a column.
  • Cytosine, uridine, uridine-2' and 3' -monophosphate disodium salt (2' -UMP and 3' -UMP), uridine-2' ,3'- cyclicmonophosphate sodium salt (cUMP) , and uridylyl (3'-»5' ) uridine ammonium salt (UpU) were purchased from Sigma (St. Louis, MO) and used without further purification. All solutions, unless otherwise stated, were prepared from a stock solution of 5.0 mM N- (2-hydroxyethyl) piperazine-N' -ethanesulfonic acid
  • HEPES HEPPES
  • Milli-Q purified water adjusted to pH 7.0. Solutions were stored and reactions conducted in RNAse free plastic vials further sterilized by heating at 120°C for 20 minutes in an autoclave. All kinetic runs were thermostated at 37°C in a water bath.
  • High-performance liquid chromatography was performed on a Waters 501 equipped with a Waters model 440 absorbance detector, monitoring at 254 nm.
  • a YMC, Inc., USA ODS-AQ column (150 mm x 4.6 mm I.D.) was used. Satisfactory separation was achieved with an isocratic gradient (10 mM NaH 2 P0 4 adjusted to pH 5.6 with 1% methanol) with a flow rate of 1.0 ml/min.
  • a Beckman DU-7 spectrometer was used to confirm the concentrations of EuB2T2 txp.
  • the reaction solutions were prepared by diluting 100 ⁇ l of UpU (2.94 mM) , 25 ⁇ l of Eu(N0 3 ) 3 (3.5 ⁇ m) , and 100 ⁇ l of cytosine (0.423 mM) , as internal standard, in 375 ⁇ l of 5.0 mM HEPES solution.
  • the reactions were carried out as for EuB2T2 txp.
  • the reaction solutions were prepared by diluting 100 ⁇ l of UpU (2.94 mM) , 50 ⁇ l of EuB2T2 txp (7.8 mM) , and 100 ⁇ l of cytosine (0.423 mM) , as internal standard, in 350 ⁇ l of 5.0 mM HEPES solution.
  • the rate of UpU hydrolysis was monitored by removing 15 ⁇ l aliquots which were frozen until HPLC analysis was possible. All samples were microfiltered (0.2 ⁇ m) prior to injection on the HPLC.
  • Uridine-2' -monophosphate, uridine-3' -monophosphate, and uridine-2' :3' -cyclicmonophosphate (cUMP) were also observed by HPLC; this indicates a hydrolytic rather than an oxidative mechanism for the cleavage reaction.
  • Uridine-2' :3' -cyclicmonophosphate reached a steady state concentration, implying that the texaphyrin complex hydrolyzed cUMP as well.
  • a 0.15 mM aqueous solution of Eu(N0 3 ) 3 has a pseudo-zero order rate constant of 2.2 ⁇ 0.35) x 10 "4 mM/h.
  • the Eu(III) complex of HAM displayed a pseudo-zero order rate constant of 4.1 x 10 "4 mM/h.
  • the texaphyrin complex is found to be more effective than the HAM system.
  • Lu(III) 1.91 x 10 "4 a The concentrations of the Lanthanide (III)B2T2 txph(N0 3 ) 2 are all approximately 0.25 mM. Further evidence supporting the catalytic effect of the texaphyrin metal complex was obtained by monitoring the formation of uridine produced from the Eu(T2B2Txp) (II) catalyzed decomposition of uridine- 2' ,3' -cyclicmonophosphate (cUMP) .
  • RNA transcripts from an isolated clone was the homogenous RNA substrate.
  • the transcripts and their degradation products were visualized by polyacrylamide gel electrophoresis and autoradiography.
  • a polymer-supported europium texaphyrin would be created and used as described hereinbelow for the unsupported complex. It is contemplated that the polymer-supported version would function essentially as the free version, but with the significant added advantages associated with being attached to a column, e.g., the purification of the hydrolysed products.
  • pGEM ® -3Z vector and Riboprobe ® RNA transcript systems were obtained from Promega Corporation, Madison, Wisconsin.
  • a 4.3 kb fragment of the mouse lb Multi Drug Resistant gene (MDR) was cloned into the EcoRI site of the pGem 3Z vector and its orientation determined.
  • the plasmid was used in transcription reactions and when digested with BamHI, T7 RNA polymerase makes a transcript from this template that is approximately 2000 bases long.
  • the transcription reaction consisted of 100 ng of Ba HI digested pGem 3Z/4.3 MDR#3, 20 ⁇ l of 5X transcription buffer, triphosphate nucleotides (A,C,G) at 500 ⁇ M, UTP at 100 ⁇ M, 50 ⁇ C of 32 -P ⁇ f-UTP (3000 Ci/mmol) , 10 mmol DTT, 120 units of RNasin and 70-100 units T7 RNA polymerase. This reaction was brought up to a total volume of 100 ⁇ l with DEPC treated double distilled water. The reaction was allowed to incubate at 37° C for 1.5 hours.
  • the entire reaction volume was then run over a G-50 Sephadex column (Nick column, Pharmacia) pre- equilibrated with 20 mM Tris pH 7.0, 2 mM EDTA, 0.1% SDS.
  • the transcript was eluted from the column in the second 400 ⁇ l volume applied to the column. Any unincorporated nucleotide was left on the column.
  • EuB2T2 txph is able to hydrolyze RNA substrates. Since the texaphyrins have such versatility for functionalization, this result has significant implications for the construction of site-specific cleaving reagents for nucleic acids.
  • the present section provides antisense agents using a texaphyrin metal complex-oligonucleotide conjugate that effects the hydrolysis of its RNA complement without the participation of endogenous nucleases.
  • a DNA-EuTx- oligonucleotide conjugate was synthesized based on the functionalized texaphyrin.
  • This "ribozyme analogue” provides an example of oligodeoxynucleotide-directed, metal catalyzed hydrolysis of a complementary RNA oligomer.
  • Polymer-supported DNA-EuTx-oligonucleotide conjugates, created as described above, could be used as described for the unsupported complex. It is contemplated that the polymer-supported version would function essentially as the free version, but with all the advantages associated with being attached to a column, such as the purification of the hydrolysed products.
  • oligonucleotides Two 20-mer oligonucleotides were machine-synthesized to contain alkylamine groups at either the 5-position of an internal thymine residue or the 5' -end terminal phosphate. Oligodeoxynucleotide-amines modified on the 5-position of thymine were purchased from Oligo's Etc. (Wilsonville, Oregon) ; oligodeoxynucleotide-amines modified on the 5' end were purchased from Keystone Laboratories, Inc. (Menlo Park, California) . Oligonucleotides were HPLC purified and precipitated using LiCl prior to use.
  • RNA 30-mer was obtained as substrate (Keystone Labs, Inc., Menlo Park, California), with a sequence selected from a unique site within the gene transcript for multiple drug resistance. Sequence is complementary at 1562 bases post-transcriptional start site in mouse multidrug resistance protein mRNA.
  • the 3' - 32 P-labelled substrate was incubated with an excess of oligodeoxynucleotide conjugate at 37°C for 18-24 h in a buffered salt solution, ethanol precipitated, and assayed on a 20% denaturing polyacrylamide gel. About 30% cleavage occurred near the expected location of the europium (III) texaphyrin complex upon hybridization with the conjugate.
  • Cleavage yield was measured by densitometry and calculated as ratio of cleavage band to intact material. The corresponding cleavage bands were not observed when this same substrate was incubated with oligonucleotides that were non-complementary in sequence, unmodified, or were modified internally with the complex. Control reactions indicate that ambient light, calf thymus DNA or type of buffer (Tris acetate or HEPES, EDTA, pH 6.0-8.0) had no apparent effect on cleavage efficiency. EDTA inhibits cleavage by free lanthanide (III) cations.
  • the cleavage fragments co-migrate with bands in sequencing lanes produced by incubation of substrate under alkaline conditions or subjected to partial digestion with a series of base-specific ribonucleases. This observation is consistent with a hydrolytic mechanism, presumably involving the EuTx acting as a Lewis acid that facilitates an intramolecular attack of the 2'-hydroxyl group to effect cleavage. There are bands indicating site-specific cleavage of the ribonucleotide target sequence in the absence of any added cleavage reagents.
  • the selectivity of the texaphyrin complexes is enhanced by covalently linking oligonucleotides onto the periphery of the macrocycle. Since the metal complexes do cleave RNA over DNA preferentially, the DNA appendages would remain intact during the hydrolysis. The DNA arm will recognize and bind to an appropriate RNA segment, effectively increasing the metal concentration at these loci relative to the overall metal concentration in solution. Phosphate ester hydrolysis will therefore be significantly increased at specific locations along the RNA backbone.
  • primers (known or deduced) for PCR could be coupled to a hydrolytic divalent or trivalent texaphyrin complex to induce hydrolysis of proximal RNA or DNA.
  • Matrix-supported texaphyrin complexes that bind to complementary RNA or DNA sequences via an appended oligonucleotide could be employed to cleave RNA or DNA proximal to this specific site. Either one or two texaphyrin molecules may be attached to the DNA.
  • solid-supported catalysts can also be developed from the homogeneous hydrolysis catalysts described in each of the sections B through D.
  • the advantages of the new solid-supported catalysts will include ease of separation of catalyst from substrate and product as well as the development of continuous flow processes.
  • the present section provides for the site-specific light-dependent cleavage of DNA by lutetium(III) texaphyrin-oligonucleotide conjugate.
  • a reaction mixture was prepared by adding ca. 300,000 cpm of 5' - 32 P-labeled DNA 36-mer (4 ⁇ L) to a solution made from lutetium(III) texaphyrin- oligonucleotide conjugate (2.5 ⁇ L, 407 nM stock solution, R 8 of the texaphyrin was NH- (CH 2 ) 6 -P0 4 -oligonucleotide) , 4X buffer (5 ⁇ L) and water (8.5 ⁇ L) to produce a final volume of 20 ⁇ L.
  • the oligonucleotide portion of the conjugate was complementary to a region of the DNA 36- mer.
  • Final conjugate concentration was 50 nM.
  • the 4X buffer is 400 mM NaCl, 200 mM HEPES, pH 7.5, 100 ⁇ M EDTA.
  • Eight reaction mixtures were pipetted into O-ring type Eppendorf tubes (1.6 mL) .
  • Two additional reaction mixtures (tubes 1 and 6) were prepared in the same way, except that an equal volume of water was substituted for the LuTx-DNA conjugate.
  • Tubes 1-5 were covered with an atmosphere of oxygen, and tubes 6-10 with an atmosphere of argon. Samples were sealed with parafilm, vortexed and centrifuged briefly, and then irradiated with laser light via the side of the Eppendorf tube. The laser was set at 752 nm and a power density of ca. 150 mW/cm 2 was used (ca.
  • Control reactions containing free lutetium(III) B2T2 texaphyrin were prepared by adding ca. 300,000 cpm of 5'- 32 P-labeled DNA 36-mer (4 ⁇ L) to a solution made from lutetium(III) texaphyrin B2T2 (U.S. Patent 5,252,720, incorporated by reference herein) (5 ⁇ L, 2 ⁇ M stock solution) , 4X buffer (5 ⁇ L) and water (6 ⁇ L) to produce a final volume of 20 ⁇ L.
  • Final LuB2T2Tx complex concentration was 500 nM.
  • Eight reaction mixtures were pipetted into 0-ring type Eppendorf tubes (1.6 mL) .
  • Tubes 11 and 16 Two additional reaction mixtures (tubes 11 and 16) were prepared in the same way, except that an equal volume of water was substituted for the LuB2T2Tx solution. Tubes 11-15 were covered with an atmosphere of oxygen, and tubes 16-20 with an atmosphere of argon. Samples were irradiated, ethanol precipitated, and analyzed by electrophoresis as described above.
  • the resulting autoradiograph indicated substantial cleavage only in those lanes that contained the 12-mer LuTx-DNA conjugate.
  • the cleavage sites covered four residues, proximal to the anticipated location of the
  • LuTx-DNA conjugate Both the locations of cleavage and the much greater efficiency of conjugate cleavage relative to that caused by free complex are consistent with a model whereby hybridization of the DNA increases the local concentration of the LuTx and effects site- specific cleavage.
  • the autoradiograph also contained information regarding cleavage mechanism:
  • the presence of oxygen in reactions 2-5 clearly increased the efficiency of DNA strand breakage. That cleavage occurred at all in samples under argon is presumably attributable either to ambient light prior to the layering with argon, or else to incomplete replacement of the atmosphere in these tubes.
  • the positive effect of oxygen on cleavage implicates singlet oxygen or other oxygen product as the intermediary species responsible for DNA strand breakage.
  • the present section provides for the site-specific light-dependent cleavage of DNA by lutetium(III) texaphyrin-2' -0-methyl RNA oligonucleotide conjugates.
  • Reaction mixtures were prepared by adding ca.
  • the 4X buffer is 400 mM NaCl, 200 mM HEPES, pH 7.5, 100 ⁇ M EDTA.
  • Two conjugate-free controls (samples 1 and 8) were prepared by substituting water for conjugate solution. Samples 1, 4-8, and 11-14 were irradiated for 4.5 hours at 37°C using a 75 watt incandescent light at ca. 9 inches above the heating block. Samples 2, 3, 9, and 10 were incubated without exposure to light at 37°C. The DNA was precipitated with ethanol using standard methods following incubation.
  • Samples 6, 7, 13, and 14 were dissolved in 10% aqueous piperidine solution (50 ⁇ L) , heated at 90°C for 30 minutes, then freeze-dried. All samples were resuspended in 50% formamide loading buffer, denatured at 90°C for 5' and analyzed by electrophoresis on a 20% denaturing polyacrylamide gel.
  • the resulting autoradiograph indicated substantial cleavage only in those lanes that contained the appropriate complementary 15-mer LuTxp 2'-0-methyl RNA conjugate.
  • the cleavage sites covered three to four residues, proximal to the anticipated location of the LuTxp complex. These cleavages are consistent with a model whereby hybridization of the 2' -O-methyl-LuTxp conjugates to their complementary sequences of DNA increases the local concentration of the LuTxp and effects site-specific cleavage.
  • the autoradiograph also contained information regarding cleavage mechanism: Certain positions within the cleavage site are clearly more reactive to cleavage than others. Definitive identification of these more reactive bases awaits further experimentation, but are tentatively assigned to positions containing purine bases.
  • the maximal extent of cleavage observed was roughly 10%, and was obtained using a piperidine treatment of the light-exposed samples.
  • the effect of this piperidine treatment is at least a 10-fold increase in cleavage products, indicating that initial DNA lesions formed by the photochemical reaction require base assistance to efficiently produce strand breaks.
  • As the extent of light-induced cleavage in non-piperidine-treated lanes is far lower than that obtained using laser irradiation, it may be possible to observe an increase in the yield of cleavage products by using both laser and piperidine treatments.
  • a texaphyrin-oligonucleotide conjugate of a derivatized RNA such as the 2'-0-methyl RNA analog used herein may provide stability against self-cleavage.
  • RNA is hydrolyzed by LuTxp, however, the 2'-O-Me RNA lacks a 2'-OH and, therefore, is stable to hydrolysis. Therefore, an RNA analog oligomer may be more stable than a DNA oligomer for the Txp-oligonucleotide conjugate.
  • the synthesis of RNA analog-conjugates is the same as for Txp-DNA conjugates discussed previously herein.
  • An RNA- analog conjugate may be complementary to an antisense or a sense strand of DNA and forms a triple helix in the process of binding to a double helix.
  • a polymeric texaphyrin itself may be employed as the texaphyrin species.
  • This Example describes the generation of polymeric texaphyrins, as may - Ill - be used as part of a polymer-supported construct, or indeed, in other embodiments in which their polymeric nature would be an advantage, such as those outlined below.
  • Examples 9 and 10 of co-pending application, PCT publication WO 94/09003, are specifically incorporated herein by reference for the purposes of supplementing the present example in terms of describing the synthetic methodology underlying the generation of polymeric species of expanded porphyrins.
  • a variety of texaphyrin derivatives are obtainable via alkylation of the phenolic precursors prior to cyclization and metallation. Some of these derivatives can be treated as components in polymer forming reactions. For instance, a T2Bl-type derivative may be elaborated synthetically to provide a diamine substituent that may be polymerized by condensation with any of a variety of activated diesters, such as that of terephthalic acid as follows.
  • R7 OH,OCHCH2OH,O(CH2CH2 ⁇ ) n CH3
  • T2B2-type derivative could be elaborated to bear two monoamine substituents, that may then be polymerized in a similar fashion.
  • Addition polymers may also be prepared. Alkylation of phenolic precursors with allylic or polyene halides would, upon elaboration, produce texaphyrin derivatives having alkene or polyene substituents. These compounds would serve as components of addition polymerization-type reactions with other alkenes, dienes, or polyenes that may or may not contain a texaphyrin as follows.
  • R7 OH, OCH2 CH2OH, O(CH2CH2 ⁇ ) n CH 3
  • the properties of the resulting polymeric materials will depend on the identity of the metal cation within the macrocycle, substituents on the macrocyclic rim, the identity of the copolymer, and the degree and type of polymerization. These properties in turn serve to define the application for which the polymer may be used.
  • a gadolinium(III) texaphyrin with water solubilizing substituents polymerized with a hydrophilic partner to form a copolymer with an average molecular weight of 10-20 kiloDalton is contemplated to serve as a useful MRI blood pool agent.
  • polymerization of more hydrophobic components is contemplated to produce materials suitable for molding and shaping.
  • Wires drawn from such a material might find application in biological implants, such as catheters, which could be visualized by MRI.
  • Such materials could also conceivably find application as semiconducting components, due to the organometallic nature of these materials, or linear optical devices.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

Abstract

L'invention porte sur différentes téxaphyrines nouvelles sur matrice support dont la matrice support polymère ou solide, est modifiée par covalence par l'adjonction d'une ou plusieurs téxaphyrines ou leurs dérivés; sur des procédés d'obtention de téxaphyrines à support polymère, dont les supports chromatographiques; sur des dispositifs tels que des cathéters pouvant servir, par exemple, à la séparation d'éléments neutres ou anioniques et dans des applications relatives à l'hydrolyse des esters phosphoriques ou à des procédés catalytiques, à l'IRM et à des TPD.
EP95920377A 1994-04-28 1995-04-28 Supports solides de la texaphyrine et dispositifs associes Withdrawn EP0758250A1 (fr)

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US5714328A (en) * 1995-06-07 1998-02-03 Board Of Regents, The University Of Texas System RNA photocleavage using texaphyrins
US6022959A (en) * 1996-08-20 2000-02-08 Pharmacyclics, Inc. Nucleic acids internally-derivatized with a texaphyrin metal complex and uses thereof
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WO1995029702A1 (fr) 1995-11-09

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