WO2003055887A1

WO2003055887A1 - Conjugated porphyrin, chlorin or bacteriochlorin chromophore

Info

Publication number: WO2003055887A1
Application number: PCT/GB2002/005867
Authority: WO
Inventors: Ross William Boyle; John Greenman
Original assignee: Wellcome Trust Limited
Priority date: 2001-12-21
Filing date: 2002-12-20
Publication date: 2003-07-10
Also published as: AU2002353209A1; GB0130778D0

Abstract

The invention provides a porphyrin, chlorin or bacteriochlorin chromophore, which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein so as to enable delivery of the chromophore to a selected biological target in vitro or in vivo, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is such that either on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 5% or less; or, where said protein is bovine serum albumin, on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 20% or less. The invention further provides methods for the synthesis of such chromophores, and methods for the use of such chromophores in therapy and in analysis.

Description

CONJUGATED PORPHYRIN, CHLORIN OR BACTERIOCH ORIN CHROMOPHORE

The present invention relates to novel poφhyrin and porphyrin-based chromophores, which may be particularly useful in a range of photodynamic applications, including photochemotherapy and fluorescence analysis and imaging.

The importance of poφhyrin and poφhyrin-based chromophores both as research tools, for example in fluorescence-activated cell sorting (FACS), and as therapeutic agents in photodynamic therapy (PDT) for bringing about the death of undesirable cells in vivo, is wic'ely recognised in the art. Each of these applications is dependent on the ability of the chromophore to be excited by incident light to a singlet excited state, and to decay to a lower energy state with the consequent emission of energy. This energy may be emitted in the form of fluorescent light at a specific wavelength, thereby enabling a cell or biostructure attached to the decaying chromophore to be visualised, and/or sorted by FACS. Alternatively, the energy of excitation may be dissipated by initial conversion of the singlet chromophore into the triplet excited state, followed by the transfer of energy to another triplet such as dioxygen, with the consequent formation of singlet oxygen. Singlet oxygen is a powerful cytotoxic agent, and hence where this latter process occurs in or in the immediate vicinity of a cell, it will usually result in the death of that cell. Accordingly, the chromophore can be exploited both for its fluorescent properties, and for its ability to act as a photosensitiser.

It is evident that for the puφoses of both fluorescence imaging/analysis and PDT, some degree of control over the localisation of the chromophore in vitro or in vivo is a prerequisite. This is particularly important in photodynamic therapy, as the typical sphere of radiation of singlet oxygen produced by decay of a chromophore is no more than O.lμm in diameter, so that in order to bring about the death of a target cell, the chromophore must usually be positioned immediately alongside, or preferably within, that cell.

Efforts have been made to achieve the specific targeting and/or attachment of poφhyrin chromophores to biological targets in vitro, in particular for the puφoses of FACS and fluorescence imaging, by covalently conjugating the chromophores to suitable protein delivery molecules. This approach has however given rise to various difficulties. In particular, problems have arisen because of the tendency of poφhyrins to bind non- covalently to protein molecules in aqueous conditions, such as during the conjugation step. Such non-covalent binding is primarily attributable to the hydrophobic character of poφhyrin molecules, which promotes association with hydrophobic portions of polypeptides in the presence of water. Not only does non-covalent binding between the poφhyrin and protein delivery molecules reduce the yield of covalently bound poφhyrin, but this effect can also hinder the subsequent targeting process. Upon introduction of the protein delivery molecules to a cell or protein mixture, non-covalently bound poφhyrin can be readily and non-specifically transferred to non-target cell surfaces or proteins, thus destroying the specificity of the targeting process.

Typically, the problem of non-covalent protein/poφhyrin binding is alleviated by way of an electroelution purification step carried out on the protein/poφhyrin mixture after the conjugation step, by which any poφhyrin non-covalently bound to protein can be separated and discarded. However, this addition purification step is inexpedient and costly, especially when carried out on an industrial scale.

Accordingly, the provision of poφhyrin- or poφhyrin-based chromophores which can be effectively covalently conjugated to protein molecules whilst showing negligible or low levels of non-covalent protein binding, and which when so conjugated display good photodynamic activity, remains a desirable objective.

According to one aspect of the present invention therefore, there is provided a poφhyrin, chlorin or bacteriochlorin chromophore, which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein so as to enable delivery of the chromophore to a selected biological target in vitro or in vivo, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is such that on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 5% or less.

It has been found that by increasing the hydrophilicity of a poφhyrin, chlorin or bacteriochlorin chromophore by way of modification of the chromophore to include hydrophilic meso substituents in accordance with the invention, the propensity of the chromophore to bind non-covalently to proteins under standard protein conjugating conditions can be substantially reduced. Where the degree of non-covalent binding under standard protein conjugation conditions is reduced to 5% or less in accordance with the invention, it is found that adequate specificity in delivering the chromophore to said selected biological target, through introducing said protein and chromophore bound thereto to said biological target, can be achieved without the need for any further purification steps, by way of electroelution or otherwise.

Preferably, said percentage of non-covalently bound chromophore out of total protein-bound chromophore may be 4% or less; more preferably 3% or less, still more preferably 2% or less, even still more preferably 1% or less; most preferably 0-0.5%.

Suitably, said protein may be a delivery protein with specific affinity for said biological target. Thus, said delivery protein may comprise an antibody or a fragment thereof, such as a monoclonal antibody or fragment thereof, a polyclonal antibody or fragment thereof, or a single-chain Fv antibody fragment (ScFv); a ligand or ligand mimetic; or an enzyme or receptor mimetic. Where said biological target is a cell or a membrane, said delivery protein may possess specific affinity for a molecule or structure exposed on the surface of said cell or membrane. In preferred embodiments, said molecule or structure exposed on the surface of said cell or membrane may be a receptor or channel, which receptor or channel is adapted to cause or allow the passage of a molecule bound thereto across said membrane or into said cell. Thus, where said biological target is a cell, said delivery protein may be adapted to be internalised into said cell upon binding to said cell.

Alternatively, said protein may be a bridging polypeptide, which bridging polypeptide is adapted to be bound or linked to a complementary bridging polypeptide, which complementary bridging polypeptide can be bound or linked to said biological target or to a delivery protein with specific affinity for said biological target so as to enable delivery of said protein molecule to said biological target. Said bridging polypeptide may for example comprise avidin streptavidin whilst said complementary bridging polypeptide comprises biotin; or vice versa. As an alternative example, said bridging polypeptide may comprise calmodulin whilst said complementary bridging polypeptide comprises calmodulin binding peptide; or vice versa.

Suitably, said protein may be free or substantially free of specific binding sites for poφhyrin-like molecules, including poφhyrins, chlorins and bacteriochlorins.

Evidently, for any given poφhyrin, chlorin or bacteriochlorin chromophore, the degree of non-covalent protein binding may vary slightly from protein to protein, depending on the structure and properties of each protein. It has however been found that where the degree of non-covalent binding of a poφhyrin, chlorin or bacteriochlorin chromophore to bovine serum albumin (BSA) under standard protein conjugation conditions, measured in accordance with the invention, is no greater than 20% of total BSA-bound chromophore, the hydrophilicity of the chromophore will always be sufficient for use in accordance with the invention. BSA possesses a number of binding sites for poφhyrin-like molecules, which increases its affinity for poφhyrin, chlorin and bacteriochlorin chromophores. Accordingly a rate of 20% non-covalent binding between a poφhyrin, chlorin or bacteriochlorin chromophore and BSA will typically correspond to a significantly lower rate of non-covalent binding between said chromophore and a protein for effecting delivery of said chromophore to a specific biological target.

According to another aspect of the present invention, therefore, there is provided a poφhyrin, chlorin or bacteriochlorin chromophore, which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is sufficient to ensure that on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 20% or less, wherein said protein is bovine serum albumin.

In preferred embodiments of this aspect of the invention, said percentage of non- covalently bound chromophore out of total protein-bound chromophore may be 18% or less; more preferably 15% or less; still more preferably 13% or less; yet more preferably 10% or less; even still more preferably 7% or less; even yet more preferably 5% or less; most preferably 3% or less.

In preferred embodiments of the invention, a chromophore in accordance with the invention is not a 5, 15-di-me.sO-substituted poφhyrin, chlorin or bacteriochlorin chromophore or a 5,10, 15,20-tetra-mesO-substituted poφhyrin, chlorin or bacteriochlorin chromophore wherein the 5-meso substituent is a conjugating substituent as hereinbefore defined and the 15-meso substituent and as the case may be the 10- and 20-meso substituents are each the same one of the following groups:

In further preferred embodiments of the invention, a chromophore in accordance with the invention is not a 5,15-di-we-fo-substituted poφhyrin, chlorin or bacteriochlorin chromophore or a 5,10, 15,20-tetra-mesø-substituted poφhyrin, chlorin or bacteriochlorin chromophore wherein the 5-meso substituent is a conjugating substituent as hereinbefore defined and the 15 -me.ro substituent and as the case may be the 10- and 20-meso substituents are each the same one of the following groups:

Suitably, said standard protein conjugation conditions may comprise a mixture of 10% of a stock solution of said chromophore in DMSO with 90% of an aqueous solution of said protein, said mixture being buffered to pH 9 so as to ensure that all lysine residues remain uncharged.

Advantageously, said percentage of non-covalently bound chromophore out of total protein-bound chromophore may be measured by separating any unbound chromophore from said protein following said incubation, for example by passing said incubated mix of chromophore and protein down a gel filtration column to obtain said protein and any chromophore bound thereto; loading said protein and any chromophore bound thereto onto a polyacrylamide gel and carrying out SDS-PAGE, so as to separate said non-covalently bound chromophore from said protein; excising from said gel said protein and said non-covalently bound chromophore; and carrying out a spectrophotometric analysis to determine the relative amounts of said non-covalently bound chromophore and of said chromophore covalently bound to said protein, so as to enable calculation of the percentage of non-covalently bound chromophore out of total protein-bound chromophore following said incubation.

Said one, two or three hydrophilic meso substituents may comprise any hydrophilic groups which when attached to a poφhyrin, chlorin or bacteriochlorin chromophore, are capable in combination of increasing the hydrophilicity of said chromophore to a level sufficient to ensure a reduced degree of non-covalent binding of the chromophore to said protein under standard protein conjugation conditions in accordance with the invention.

In a highly preferred aspect of the invention, one or more of said hydrophilic meso substituents may comprise a charged substituent, such as a zwitterionic substituent possessing both positively and negatively charged moieties, other than:

or or a cationic substituent having a net positive charge, other than:

or an anionic substituent, having a net negative charge.

The inclusion of one or more hydrophilic meso substituents around the core of a poφhyrin, chlorin or bacteriochlorin chromophore in accordance with the invention results in enhanced solubility in basic buffer/DMSO or DMF co-solutions which are commonly used in protein bioconjugation. Increased hydrophilicity also produces a marked reduction in the tendency of the chromophore to bind non-covalently to proteins. Where the chromophore is to be conjugated to a targeting protein such as a monoclonal antibody for delivery to specific cells or tissues, for example for the puφoses of PDT or FACS, a decrease in non-covalent binding between the chromophore and the protein will reduce the degree of non-specific transfer of chromophore to cell surfaces, which will substantially increase the accuracy of targeting the chromophore to the cells or tissue of interest.

It has now furthermore been found that the inclusion of one, two or three zwitterionic, cationic or anionic hydrophilic me O substituents around a poφhyrin, chlorin or bacteriochlorin core, in accordance with the invention, results in a chromophore which not only benefits from the aforementioned reduction in non-covalent protein binding, but also acts as a highly effective photosensitiser. More specifically, it has been found that the presence around the periphery of a poφhyrin, chlorin or bacteriochlorin chromophore core of one, two or three zwitterionic, cationic or anionic hydrophilic meso substituents in accordance with the invention tends to result in a distinctive pattern of intracellular chromophore biodistribution. In particular, the presence of one, two or three cationic or zwitterionic substituents around a chromophore core in accordance with the invention has been found to correlate with intracellular localisation of the chromophore principally in or around the mitochondria. Localisation of a chromophore in or around the mitochondria is especially advantageous for the puφoses of photodynamic therapy, as this will enable efficient and targeted disruption of the mitochondria, thereby triggering cellular apoptosis. In the context of in vivo photodynamic therapy, cell death by apoptosis is to be preferred over cell death by necrosis, as the apoptotic process is naturally succeeded in vivo by the building of healthy tissue, thus minimising formation of scar tissue and organ damage and loss of function.

Meanwhile, the presence of one, two or three anionic substituents around a chromophore core in accordance with the invention is found to correlate with intracellular localisation of the chromophore principally in or around the lysosomes.

In some embodiments, said hydrophilic substituent may comprise a quartenised pyridyl (pyridiniumyl) ring. Where said pyridiniumyl ring comprises no charged ring substituents, the hydrophilic substituent will constitute a cationic substituent, owing to the presence of the single positive charge on the quartenised nitrogen ring atom. Where the pyridiniumyl ring comprises a single charged ring substituent which is a negatively charged ring substituent, the hydrophilic substituent will constitute a zwitterionic substituent. Where the pyridiniumyl ring comprises charged substituents which impart to the ring a net positive or a net negative charge, the hydrophilic substituent will constitute a cationic or anionic substituent respectively.

Said pyridiniumyl ring may be N-linked to said poφhyrin, chlorin or bacteriochlorin chromophore core, such that the N-linkage of said pyridiniumyl ring quartenises the nitrogen atom. Alternatively, said pyridiniumyl ring may be linked by a carbon atom in said ring to said poφhyrin, chlorin or bacteriochlorin chromophore core and may comprise one quartenising ring substituent Q which is N-linked to said pyridiniumyl ring for quartenising said nitrogen atom. Said quartenising ring substituent Q may comprise ethyl, or branched or linear propyl, butyl, pentyl, hexyl, heptyl or octyl, or aryl such as phenyl, or heteroaryl such as pyridyl. Alternatively, said quartenising ring substituent Q may comprise a hydrophilic group W as hereinafter defined. Suitably, said pyridiniumyl ring may be substituted one or more times by one or more hydrophilic groups W as hereinafter defined. Each hydrophilic group W may be a group selected from R₃L, or YιR , or (R-N-RsJx. or R YιY₂R₅, or Y|R₄Y₂R₅, or -COO^", or -SO₃ ^", or hydroxy, or oxo; wherein R₃ is methyl or ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, or phenyl, or pyridyl, or pyridiniumyl, or a group R₆R , where R₆ is methyl or ethyl and R₇ is phenyl or pyridyl or pyridiniumyl; where R₃ does not comprise pyridiniumyl, L represents one or more groups selected from -OH, -COO^" (or -COOH) or -SO₃ ^"(or SO₃H); where R₃ comprises pyridiniumyl, L represents one or more groups selected from -OH, -COO^" (or -COOH), -SO₃ ^" (or SO₃H), or hydrogen; Y, and Y₂ are independently selected from O and S; R is selected from a single bond, methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, phenyl, pyridyl, and pyridiniumyl; R₅ is selected from hydrogen, methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, phenyl, pyridyl, and pyridiniumyl; and x is an integer from 1 to 5.

Advantageously, said R₃ may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, and said L may include a terminating hydroxy, carboxy or sulfonate group. In particularly preferred embodiments therefore, said hydrophilic group W may be a group - (CH₂)_aOH, or -(CH₂)_aCOO^"(or -(CH₂)_aCOOH), or -(CH₂)_aSO₃ ^" (or -(CH₂)_aSO₃H) where a is 1, 2, 3, 4, 5, 6, 7 or 8; most preferably 1, 2 or 3. Thus, said hydrophilic group W may advantageously be hydroxymethyl, hydroxyethyl, ethylsulfonate or propylsulfonate.

Suitably, said Yi may be O, and said x may be greater than 1. Accordingly, said hydrophilic group W may comprise polyethylene glycol, preferably C₂.₃₀ polyethylene glycol.

Alternatively, said hydrophilic group W may comprise a glycosyl group. In particular, said hydrophilic group W may comprise a glycosyl group which is a sugar such as glucose, mannose, maltose, or a thiosugar such as thiogalactopyranose, thioglucopyranose or thiomannopyranose. More specifically, said hydrophilic group W may comprise one of the following glycosyl groups, where x is an integer between 1 and 6:

Alpha or beta glucopyranose

Alpha or beta mannopyranose

Alpha or beta galactopyranose

Alpha or beta maltose

Alpha or beta thioglucopyranose

Alpha or beta thiomannopyranose

Alpha or beta thiogalactopyranose

Alpha or beta thiomaltose As yet a further alternative, said hydrophilic group W may comprise an amino acid. Amino acids are readily available and are well-known and characterised. Amino acids typically have good hydrophilicity owing to their zwitterionic character. Particular amino acids which may be utilised in accordance with the invention include lysine, cysteine, tyrosine, aspartate, glutamate, serine and threonine.

Said hydrophilic group W may be further substituted one or more times by hydroxy or oxo, so as further to increase the hydrophilicity of the group. Preferably, the total number of carbon atoms in said hydrophilic group W may not exceed 30.

The presence of one or more hydrophilic groups W linked to said pyridiniumyl ring will serve to improve the hydrophilicity of the pyridiniumyl ring. Preferably, said pyridiniumyl ring may be substituted one or two times by one or two hydrophilic groups W respectively.

In other embodiments, said hydrophilic substituent may comprise a quartenised amine group -N⁺QιQ₂Q3 or a quartenised phosphonium group -P⁺QιQ₂Q3. each of which Qi, Q₂ and Q₃ is selected from methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, aryl such as phenyl, heteroaryl such as pyridyl, and a hydrophilic group W as hereinbefore defined. Optionally, said Qi, Q₂ and/or Q₃ may be substituted by a hydrophilic group W as hereinbefore defined, such as by a hydroxy, oxo, sulfonate, or carboxylate group, so as to improve the hydrophilicity of said cationic substituent.

Advantageously, at least one, more preferably two, most preferably each of said Qi, Q₂ and Q₃ may comprise an uncharged aryl or heteroaryl moiety, such as a phenyl ring, naphthyl ring, anthracene ring or a pyridyl ring. The presence of three aromatic moieties directly linked to the quartenised NT or P⁺ atom will enable effective delocalisation of the positive charge on the quartenised atom. It has been found that the inclusion around a poφhyrin, chlorin or bacteriochlorin chromophore core of one or more meso substituents comprising a cationic moiety having a delocalised positive charge, such as -N⁺QιQ₂Q₃ or -P⁺QιQ₂Q₃ where each of Qi, Q₂ and Q₃ comprises an uncharged aryl or heteroaryl moiety, results in particularly effective intracellular localisation of the chromophore in and around the mitochondria, and in particularly efficient disruption of mitochondrial function on excitation of the localised chromophore. Thus, such chromophores are especially preferred for use in photodynamic therapy. Said uncharged aryl or heteroaryl moiety may be substituted one or more times by one or more substituents which do not interfere with the aromatic character of said moiety, such as (CH₂)_yR₈ where y is an integer between 0 and 6 and R₈ is hydroxy, halo, sulfonate or carboxylate.

In yet other embodiments, said hydrophilic substituent may comprise a phosphate group -P(O)(OR₇XO^") or a phosphonate group -OP(O)(OR₇)(O^"), wherein said R₇ is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl. Optionally, said negatively charged phosphate or phosphonate group may be associated with a counterion such as a sodium or potassium counterion. Alternatively, said phosphate or phosphonate group may comprise a group -P(O)(OR₇)(OH) or a group -OP(O)(OR₇)(OH).

Said pyridiniumyl ring, quartenised amine group, quartenised phosphonium group, phosphate group or said phosphonate group may be linked to the core of said poφhyrin, chlorin or bacteriochlorin chromophore by way of a linking group L₂, which linking group L-₂may comprise a group -R_\- or -R]R₂-, where each of Ri and R₂ is independently selected from a single bond, or methyl, or phenyl, or branched or linear ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, optionally substituted one or more times by one or more hydrophilic substituents W as hereinbefore defined. Alternatively, said linking group L₂ may comprise an ether or thioether chain such as a chain -A]R| A₂R₂-, where each of Ai and A₂ is independently selected from a single bond or S or O, and Ri and R₂ are as hereinabove defined, or a polyether or polythioether chain based on repeating units of said

such as a C2-₃₀ polyethylene glycol chain.

In some preferred embodiments, particularly where said hydrophilic substituent comprises a quartenised amine group

or a quartenised phosphonium group - P⁺QιQ2Q3 or an N-linked pyridiniumyl ring or phosphate group or phosphonate group, said linking group may be a group RιR₂ wherein said Ri is phenyl and said R₂ is methyl or ethyl or propyl. In other preferred embodiments, in particular where said hydrophilic substituent comprises a pyridiniumyl ring which is not N-linked, said linking group may be a group Ri wherein Ri is a single bond. In another advantageous aspect of the invention, said hydrophilic substituent may advantageously comprise or consist of a group R₁₁R.₁₂R₁₃. where Rπ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, R₁₂ is NH, O, S or CH₂, and R13 is hydrogen or a hydrophilic group W as hereinbefore defined. Said group RnRnR^ may advantageously be substituted one or more times by hydroxy, so as to improve the hydrophilicity of the group R11R12R.3. In particularly preferred embodiments, said Ru may be ethyl. Advantageously, said R₁₂ may be O and said R1₃ may be H, and said R1₁R₁₂R₁₃ may be further substituted one or more times by OH, such that said RnRπ π constitutes a polyhydroxyalkyl, preferably a dihydroxyalkyl. In other preferred embodiments, said Rι₃ may be selected from polyethylene glycol; glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose; lysine; cysteine; tyrosine; aspartate; glutamate; serine and threonine.

Suitably, said conjugating meso substituent may comprise an aryl moiety Rι₀ which is linked to said conjugating group Z. Said aryl moiety Rι₀may advantageously comprise a phenyl ring, which phenyl ring may preferably be linked by a single bond to the macrocyclic core of said chromophore or may alternatively be linked thereto by a linking group L₂ as hereinbefore defined. Advantageously, said conjugating group Z may be linked to said phenyl ring at the para (4') position thereof.

Said conjugating group Z may comprise a group which is capable of bonding covalently to an amine group on a polypeptide molecule; such as an isocyanate, isothiocyanate, or NHS ester group. Advantageously, therefore, each of the meso substituents around said poφhyrin, chlorin or bacteriochlorin should comprise no -NH-, - NH₂, -NH2⁺- or -NH₃ ⁺ groups which could become covalently bonded to said conjugating group Z. This will serve to reduce the probability of internal cross-linkage within said chromophore. Said conjugating group Z may alternatively comprise any other protein conjugating group, such as -NH₂, -NH(Cι-₆ alkyl), maleamide, iodoacetamide, ketone or aldehyde. Methods for achieving the conjugation of such groups to protein molecules are known in the art.

In especially preferred embodiments, said conjugating group Z comprises an isothiocyanato group. Isothiocyanates react readily with lysine residues to produce a stable linkage to proteins, and hence are particularly suitable for bioconjugation of chromophores in accordance with the invention.

Said conjugating group Z may be linked directly to said aryl moiety Rio by a single bond. Alternatively, said conjugating group Z may be linked to said aryl moiety Rio by a linking moiety having a relatively high degree of inflexibility and/or steric hindrance. Said linking moiety may, for example, comprise a chain of fused or linked cycloalkyl and/or cycloaryl ring structures having a total molecular weight no greater than lOOOgmol^"1. In particular, said linking moiety may comprise an anthracene, acridine, anthranil, napiithyl or naphthalene moiety, or a polyacetylene, phenylacetylene, or polyphenylacetylene moiety. When said chromophore is conjugated by said conjugating group Z to a polypeptide molecule, therefore, said linking moiety can serve to keep the photoactive core of said chromophore apart from said polypeptide, thereby helping to reduce the degree of fluorescence quenching which may be caused by said polypeptide when said chromophore is caused to fluoresce. Said linking moiety may include a linbking group L₂ as hereinbefore defined. Preferably, said linking moiety may be substituted one or more times by one or more hydrophilic substituents W as hereinbefore defined, or may include one or more ether or thioether groups -A1R1A2R₂- as hereinbefore defined. This will help to ensure that the hydrophilicity of the chromophore is not impaired by the presence of said linking moiety.

Optionally, said aryl moiety Rio may be further substituted by one or more hydrophilic substituents, such as hydroxy or oxo, which will serve to improve the hydrophilicity of said chromophore.

It will be appreciated that any of the above-defined groups, substituents or moieties may be further substituted by one or more inert atoms or groupings, such as methyl, ethyl or halo, particularly fluoro, which will not disrupt or substantially affect the properties and functionality of said groups, substituents or moieties. Furthermore, any of the above-defined groups, substituents or moieties may be further substituted one or more times by hydroxy or oxo, so as further to improve hydrophilicity. Preferably, the total molecular weight of each meso substituent will not exceed lOOOgmol^"1; more preferably the total molecular weight of each meso substituent will not exceed 700gmol^"1; still more preferably the total molecular weight of each meso substituent will not exceed SOOgmol^"1; even still more preferably the total molecular weight of each meso substituent will not exceed 300gmor'.

Preferably, a chromophore in accordance with the invention may be a poφhyrin chromophore of formula (I) below:

or a chlorin chromophore of any of formulas (II), (III), (IV), or (V) below:

(II) (III)

or a bacteriochlorin chromophore of any of formulas (VI) and (VII) below:

(VI) (VII)

wherein Rio is or comprises a conjugating meso substituent as hereinbefore described; at least one of R₂o, R₃₀ and R₄₀ is or comprises a hydrophilic meso substituent as hereinbefore defined; and each of Xi, X₂, X₃ and X is independently selected from H, OH, halogen, C₁-₃ alkyl and OC₁.₃ alkyl, or X] and X₂ and/or X₃ and X₄ together form a bridging moiety selected from O, CH₂, CH C₁.₃ alkyl, or C(Cι-₃ alkyl)₂, such that Xi and X2 and/or X₃ and X with the adjacent C-C bond form an epoxide or cyclopropanyl structure.

In some embodiments, each or some of X_\- X is H. In particularly preferred embodiments, however, each of Xi - X is OH. Accordingly, said chromophore may be a dihydroxychlorin of formula (II), (III), (IV) or (V) above or a tetrahydroxybacteriochlorin of formula (VI) or (VII) above. The hydrophilicity of dihydroxychlorins and tetrahydroxybacteriochlorins is found to be greater than that of the corresponding poφhyrins, owing to the presence of extra hydrophilic hydroxy groups around the core of the chromophore.

In some embodiments, said poφhyrin, chlorin or bacteriochlorin chromophore will comprise three hydrophilic meso substituents R20. R3o. R ₀ and one conjugating meso substituent Rι₀, such that the chromophore is tetra-meso substituted. Said three hydrophilic meso substituents R₂o, R30. R^ may be identical one to each other; or may be different one from another. In some embodiments, two of said three hydrophilic meso substituents R₂₀. R30. R-to may comprise the same group RπRι₂Rι₃ as hereinbefore defined, whilst the remaining one of said three hydrophilic meso substituents may comprise a different hydrophilic substituent in accordance with the invention.

The inclusion of three hydrophilic meso substituents together with one conjugating meso substituent enables a high degree of hydrophilicity to be imparted to the chromophore. However, the presence of such substituents can hinder the conversion of poφhyrins to chlorins and of chlorins to bacteriochlorins within the scope of the invention by way of addition reaction across the exocyclic double bonds around the chromophore core.

Accordingly, in an alternative preferred aspect of the invention, said R₃₀ may be a hydrophilic meso substituent as hereinbefore defined, whilst said R₂₀ and R ₀ may each be hydrogen, such that said chromophore is 5,15-di-me->ø-substituted.

In some embodiments, each of said R₂o and said R o is a hydrophilic group R11R12R1₃ as hereinbefore defined, and said R₃₀ is a group having a total molecular weight less than lOOOgmol^"1, preferably less than 700gmol^"\ more preferably less than 500gmol^" . Suitably, said R₃0 may comprise a phenyl ring, which ring may be substituted one or more times by one or more substituents selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, phenyl, pyridyl, or a hydrophilic group W as hereinbefore defined. Said phenyl ring may be linked directly to the macrocyclic core of said chromophore by way of a single bond, or may be linked thereto by way of a linking group which may comprise methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, phenyl, phenylmethyl, phenylethyl, pyridyl, pyridylmethyl or pyridylethyl. In preferred embodiments, said R₃0 may comprise a phenyl ring which is linked directly to the macrocyclic core of said chromophore by way of a single bond, and which is para- substituted once by methyl, ethyl, propyl, carboxylate, ethanoate, propanoate, butanoate, sulfonate, methylsulfonate, ethylsulfonate, propylsulfonate, or glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose.

Chromophores in accordance with the invention wherein R₂₀, R30. and R-t₀ are identical one to the other may be synthesised in accordance with methods known in the art, for example by acid catalysed condensation of benzaldehydes with pyrrole, or by means of the "MacDonald 2+2" method for synthesising poφhyrins from dipyrromethanes (Arsenault et al, I. Chem. Soc. 1960, 82:4384-4389 - incoφorated herein by reference).

A generalised scheme for the synthesis of 5-isothiocyanatophenyl-15- pyridiniumyl poφhyrins, chlorins and bacteriochlorins in accordance with the present invention is set out as Scheme 1 below, in which "RX" represents a quartenising group selected from CH₂CH₂COOH, CH₂CH₂OH, CH₂CH₂S0₃H and glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose:

Scheme 1

A generalised scheme for the synthesis of 5-isothiocyanatophenyl-15- methylphosphoniumphenyl poφhyrins, chlorins and bacteriochlorins in accordance with the present invention is set out as Scheme 2 below, wherein R represents ethyl, PhS0₃H, PhCOOH, or PhOH :

P^ (i) CBi PPr

(II) Rs (111) TMSI

Scheme 2

Poφhyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein at least one of said hydrophilic meso substituents comprises phenylmethylpyridiniumyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 3, wherein "R" represents 3,5-(COOH)₂, 4- CH₂CH₂SO₃H, or 2,6-(CH₂OH)₂:

Scheme 3

Poφhyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein at least one of said hydrophilic meso substituents comprises alkylphosphonatophenyl or alkylphosphatophenyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 4, wherein "R" represents OH, ONa, or 0(Cι-₆ alkyl):

Scheme 4

A generalised scheme for the production of 5-(4-isothiocyanatophenyl)- 10,20- bis(l,2-dihydroxyethyl) poφhyrin, chlorin and bacteriochlorin is set out in Scheme 5 below, where X represents C or N and R represents CH₂CH₂COOH, CH₂CH₂OH, CH₂CH₂SO₃H, or glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose; or (where X is C) methyl:

Scheme 5

A generalised scheme for the production of 5-(4-isothiocyanatophenyl)-10,20- bis(l,2-disubstituted ethyl) poφhyrin, chlorin and bacteriochlorin is set out in Scheme 6 below, where X represents N, O, S or C; Z represents N or C; R represents polyethylene glycol, glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose, lysine, cysteine, tyrosine, aspartate, glutamate, serine or threonine; and R¹ represents Me, CH₂CH₂COOH, CH₂CH₂OH, CH₂CH₂S0₃H or glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose:

X = N,0,S,C, Z = N,C, R = PEG, glycosyl, lysine, cysteine, tyrosine, aspartic acid, glutamic acid, senne, threonine, R¹ = Me, C4>CH₂COOH, C4-CH..OH, ChfeCHsSOsH, glycosyl

According to a further aspect of the present invention, there is provided a method for synthesising a sugar-substituted meso-aτy\ poφhyrin/chlorin/bacteriochlorin, comprising the steps of providing a meso-aτyl poφhyrin/chlorin/bacteriochlorin in which the meso- aryl substituent is substituted with a leaving group and at least one electron withdrawing group, and reacting this poφhyrin/chlorin/bacteriochlorin with a nucleophilic sugar such as to displace the leaving group on the meso-ary\ substituent by way of a nucleophilic substitution reaction.

It has been found that the synthesis of sugar-substituted meso-aryl poφhyrins/chlorins/bacteriochlorins by way of a nucleophilic substitution reaction, in accordance with the invention, results in improved yield.

Preferably, said meso-ary\ substituent may be a phenyl ring or a pyridyl ring. Said poφhyrin, chlorin or bacteriochlorin may comprise one, two, three or four me-jo-aryl substituents, some or each of which may be substituted with a leaving group and at least one electron withdrawing group in accordance with the invention.

As will be appreciated by the skilled man, said nucleophilic sugar acts as a nucleophile in displacing said leaving group from said meso-αry] substituent. Said nucleophilic sugar may be a deprotonated sugar such as deprotonated glucose, mannose, or maltose, having an anomeric hydroxyl group, which anomeric hydroxyl group is deprotonated. Alternatively, said nucleophilic sugar may be a thiosugar, such as thiogalactopyranose, thioglucopyranose or thiomannopyranose.

Electron withdrawing groups suitable for use in the method of the invention are known in the art. Examples include fluoro, nitro and cyano groups. In preferred embodiments, said aryl substituent may be substituted by more than one electron withdrawing group, such as by 2, 3, 4, or 5 fluoro, nitro and/or cyano groups. This will improve the rate of the nucleophilic substitution reaction. In most preferred embodiments, said aryl substituent may be substituted by a plurality of fluoros, such as by 3 or 5 fluoros. The presence of a plurality of fluoros around said meso-aryl substituent will have a minimal effect on the properties and functions of said poφhyrin, chlorin or bacteriochlorin. Leaving groups suitable for use in the method of the invention are also known in the art. Examples include fluoro, mesylate, tosylate and triflate groups. In preferred embodiments, said aryl substituent may be para-substituted by one leaving group, such as by fluoro.

In especially preferred embodiments, said me->σ-aryl substituent may be ortho- and/or meta-substituted by a plurality of fluoros by way of electron withdrawing groups, and may be para-substituted by fluoro by way of a leaving group. Preferably, therefore, said meso-aryl substituent may be pentafluoro-substituted.

Said n αcleophilic sugar may comprise protecting groups, such as acyl protecting groups; and said method may further include the step of deprotecting said sugar, such as to remove said protecting groups. The deprotection of said sugar will reduce the solubility of said sugar-substituted poφhyrin, chlorin or bacteriochlorin in tetrahydrofuran and in dichloromethane, which are solvents used in a number of important reactions including in the formation of NCS groups. However, where said meso-aryl substituent is substituted by one or more electron-withdrawing groups and/or leaving groups which tend to enhance solubility in oxygen-containing solvents, such as by three or five fluoros, the solubility of said sugar-substituted poφhyrin, chlorin or bacteriochlorin in tetrahydrofuran and dichloromethane will remain adequate to enable said poφhyrin, chlorin or bacteriochlorin to be dissolved in such solvents, notwithstanding the deprotection of said sugar.

Thus, said method may further include the step of dissolving said sugar- substituted poφhyrin, chlorin or bacteriochlorin in tetrahydrofuran or dichloromethane, and forming an NCS group on a meso-aryl substituent of said sugar-substituted poφhyrin, chlorin or bacteriochlorin, in order to obtain a sugar-substituted NCS-linked meso-aryl poφhryin, chlorin or bacteriochlorin in accordance with the invention.

Generalised schemes for the production of di-phenyl and tetra-phenyl sugar-substituted poφhyrins in accordance with the present invention are set out in Schemes 7 and 8 below.

Scheme 7

Scheme 8

The chromophores of the invention are novel, and are each capable on excitation of emitting fluorescent light at different and substantially non-overlapping wavelengths. As indicated above, the provision of conjugating group Z enables a chromophore in accordance with the invention to be specifically targeted to a specific biological target, thus facilitating control over the localisation of the chromophore in vitro or in vivo. Chromophores in accordance with the invention may therefore be usefully employed in fluorescence analysis and imaging applications (including FACS), or in PDT.

According to another aspect of the present invention, there is provided a set of fluorochromic markers for multicolour fluorochromic analysis, comprising at least two chromophores selected from the group consisting of a poφhyrin chromophore, a chlorin chromophore and a bacteriochlorin chromophore, each of which chromophores is a chromophore in accordance with the invention and each of which chromophores comprises the same 5, 10, 15 and 20 meso substituents.

It has been found that each of the chromophores in a set in accordance with the present invention will on excitation emit fluorescent light at a different discrete wavelength. Thus, all of the chromophores within the set can be excited by a single laser, producing separate emission bands which can be substantially individually resolved. Moreover, all of the chromophores provided in said set share substantially the same molecular structure, and will accordingly share substantially the same biochemical and physicochemical properties, including substantially the same degree of efficiency of protein conjugation and target delivery under given conditions. Accordingly, a set of chromophores in accordance with the present invention may be usefully employed in fluorescence analysis and sorting applications, including FACS, for the convenient sorting and analysis of several types of cells or other biological targets. The components of such a set may, for example, be introduced to a mixture comprising one or more of said different specific biological targets, under conditions which will allow the delivery of each chromophore to its respective specific biological target; and said mixture may be exposed to light so as to cause said chromophores to fluoresce. A multicolour analysis may then be carried out for identifying the different emission bands produced by each chromophore, thereby permitting counting and visualisation of the location of each of the different biological targets.

In a chromophore in accordance with the present invention, or in each member of a chromophore set in accordance with the present invention, said conjugating group Z may be conjugated to a delivery protein which is adapted to bind specifically to said biological target. Alternatively, said conjugating group Z may be conjugated to a bridging polypeptide which is adapted to bind to a complementary bridging polypeptide so as to couple said chromophore to said complementary bridging polypeptide.

In some embodiments, said bridging polypeptide may be bound to said complementary bridging polypeptide, and said complementary bridging polypeptide may comprise or be coupled to or fused with a delivery protein which is adapted to bind specifically to said biological target. Accordingly, said chromophore may be covalently linked to said delivery protein by means of said bridging polypeptide and said complementary bridging polypeptide.

According to another aspect of the present invention, there is provided a kit comprising a chromophore in accordance with the present invention or a set of chromophores in accordance with the present invention, wherein said chromophore or each chromophore is conjugated to a bridging polypeptide that is adapted to bind to a complementary bridging polypeptide so as to couple the chromophore to said complementary bridging polypeptide; and a construct or plurality of constructs each of which comprises said complementary bridging polypeptide fused or coupled to a delivery protein which is adapted to bind specifically to said biological target; the arrangement being such that said chromophore or each chromophore in the kit is adapted to bind to a different construct in the kit with specificity for said specific biological target, so as to link said or each chromophore to a delivery protein with specificity for said specific biological target.

Said delivery protein may, for example, be an antibody such as a monoclonal or polyclonal antibody or a fragment thereof with specificity for a target specific molecule on the surface of said biological target. Panels of antibodies which recognise colorectal cell lines are, for example, commercially available. Alternatively, panels of antibodies against selected cell lines, such as lung cancer cell lines include COR L23, L51, L105 and L283, can be generated using the bacteriophage display library technique, as described in Hoogenboom et al, (2000) Natural and designer binding sites made by phage display technology (Immunology Today) 21(8): 371-378. This technology enables isolation of antibody fragments, such as Mab or scFv fragments, with particular binding activity. The fragments are individually displayed on the surface of phage and can be subsequently characterised and produced in quantity. This technique has been used to raise antibody fragments against human colorectal cell lines (Topping et al, (2000) Isolation of human colorectal tumour reactive antibodies using phage display technology. Int. J. Oncol. 16(1): 187-195). The murine scDv antibody MFE-23 which reacts with high specificity with the carcinoembyronic antigen (CEA/CD66e) (Begent et al, 1996 Nat. Med. 2: 979-984) is suitable for use in connection with the present invention. In particular, said antibody may be a phage antibody, that is an antibody expressed on the surface of a bacteriophage. Alternatively said delivery protein may be a protein which is adapted to bind to one or more cell surface molecules or receptors, such as a serum albumin protein. As yet a further alternative, said delivery protein may comprise a low density lipoprotein, such as a fatty acid chain, which is adapted for insertion into a cell membrane. When conjugated to a chromophore, such a lipoprotein can serve to anchor the chromophore to a cell membrane.

Said bridging polypeptide may comprise calmodulin, and said complementary bridging polypeptide may comprise calmodulin binding peptide; or vice versa. Preferably, however, said bridging polypeptide may comprise avidin or streptavidin, and said complementary bridging polypeptide may comprise biotin; or vice versa. In particular, said or each chromophore in a kit in accordance with the present invention may be conjugated to avidin, and said or each construct may comprise a biotinylated monoclonal antibody with specificity for a target specific molecule on the surface of said biological target. Accordingly, when said avidin-linked chromophore is allowed to bind said biotinylated antibody, said chromophore will become firmly linked to said antibody. Conveniently, said or each biotinylated monoclonal antibody in the kit may be selected and/or readily substituted, so as to enable said or each chromophore to be delivered to any desired biological target. Methods for the preparation of monoclonal antibodies and for the biotinylation thereof are well known in the art.

According to another aspect of the present invention, there is provided a method for attaching a chromophore in accordance with the invention or a set of chromophores in accordance with the invention to said specific biological target or targets; comprising the steps of providing a kit in accordance with the present invention, and introducing the components of said kit into the vicinity of said specific biological target or targets, under conditions suitable for enabling the binding of said or each delivery protein to said specific biological target or targets. Advantageously, the components of said kit may be allowed to associate with one another prior to introduction to said target or targets, so as to enable the bridging polypeptide conjugated to said or each chromophore to bind to a complementary bridging polypeptide provided on one of said constructs in the kit. This will ensure that said or each chromophore in the kit is linked to a delivery protein prior to introduction of said chromophore to said target or targets. Alternatively, the components of said kit may be introduced sequentially to said target or targets.

Typically, said specific biological target may be a cell or a membrane. Said specific biological target may be in vivo or in vitro (ex vivo). Said biological target may, for example, be a cancer cell, a tumour cell, a cell infected with HIV or with any other microbe or virus, a cell responsible for detrimental activity in auto-immune disease, a foreign or diseased cell, or any other such cell.

In some embodiments of the present invention, said biological target is a cell in vitro, and said target specific molecule comprises a molecule exposed on the surface of said cell, such as a polypeptide, carbohydrate, fatty acid, lipoprotein, phospholipid or other biological molecule. Preferably, said target specific molecule is specifically expressed by, or is over-expressed by, said cell. Said target specific molecule may, for example, be a T cell marker such as CD4 or CD8. Accordingly, when a chromophore in accordance with the present invention or a chromophore forming part of a set of chromophores in accordance with the present invention is attached to said cell, and said cell is illuminated so as to cause fluorescence of said chromophore, the fluorescence of the chromophore will enable said cell to be visualised and counted and/or sorted by FACS.

According to a further aspect of the present invention, therefore, there is provided a method for fluorescence-activated sorting of target cells from a mixture of cells, comprising the step of attaching to said target cells a chromophore in accordance with the invention or a set of chromophores in accordance with the invention, illuminating said mixture of cells so as to cause fluorescence of one or more of said chromophores attached to said target cells, imparting a charge to the fluorescing cells, and passing said mixture of cells through a polarised environment so as to cause or allow said charged cells to be separated from said mixture.

According to another aspect of the present invention, there is provided a method for the visualisation and/or counting of a plurality of target cells, said target cells including cells of two or three different cell types, comprising the steps of providing a chromophore set in accordance with the present invention, which chromophore set comprises two or three chromophores each of which is adapted to be delivered to a different one of said cell types; attaching said chromophores in the set to said target cells in accordance with the method of the present invention; illuminating said target cells so as to cause the emission of fluorescence by said chromophores; detecting the fluorescent emission bands produced by each of said chromophores; and optionally measuring for each of said bands the area under an emission/wavelength curve, so as to obtain a measure of the number of fluorescent cells of each respective cell type.

In other embodiments of the present invention, said target cell is a cell in vivo, such as a cancer cell, tumour cell, or an infected, foreign or diseased cell, and said target specific molecule is a target cell specific molecule which is specifically expressed by, or is over-expressed by, or is attached to, and is exposed on, the surface of said target cell; such as a target cell specific membrane protein. Accordingly, when a chromophore in accordance with the invention is delivered to said target specific molecule, said chromophore will be caused to be attached to said cell. If said cell is subsequently illuminated with light at a wavelength suitable for causing the excitation of said chromophore, said chromophore attached to said cell may be caused to be excited, and this may result in the production of singlet oxygen in the immediate vicinity of said cell, hence bringing about the death of the cell.

In especially preferred embodiments, said target cell specific molecule comprises an internalisation receptor on the surface of said cell, which intemahsation receptor is capable of binding said delivery protein and thereby mediating the internalisation of said chromophore within said cell. Accordingly, subsequent illumination of said cell with light at a wavelength suitable for causing excitation of said chromophore may result in the production of singlet oxygen within said cell, hence bringing about the death of said cell.

The present invention therefore comprehends a method for causing the death of a target cell, comprising the step of attaching a chromophore in accordance with the present invention to said cell and illuminating said cell so as to cause the production of singlet oxygen in the vicinity of said cell, thereby causing the death of the cell. Preferably, said chromophore is attached to an internalisation receptor on the surface of said cell, which internalisation receptor is capable of mediating the internalisation of said chromophore within said cell, and said cell is thereafter illuminated such as to cause the production of singlet oxygen within said cell, thereby causing the death of the cell.

Preferably, where said chromophore is adapted to be internalised within the cell, said chromophore comprises a cationic or a zwitterionic group such as a quartenised amine or pyridyl (pyridiniumyl) group, or a quartenised phosphonium group, in particular a phosphonium group wherein the phosphorous atom is directly linked to three aryl groups such as three phenyl rings. As described above, such chromophores will tend to localise in or around the mitochondria of said cell, and on production of singlet oxygen by decay of the chromophore, will produce rapid and efficient killing of the cell.

In accordance with another aspect of the invention, there is provided a method for treating a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), comprising the step of administering to a patient in need thereof an effective amount of a chromophore in accordance with the invention, which chromophore is adapted to be targeted to a target cell specific molecule on the surface of said diseased or undesired cells for attachment thereto, such that the chromophore is caused to be attached to said cells, and illuminating said cells with light so as to cause the production of singlet oxygen in the vicinity of said cells, thereby killing said cells. Suitably, said target cell specific molecule comprises an internalisation receptor, and said chromophore is adapted to be internalised within said cells on delivery to said internalisation receptor, such as to enable the production of singlet oxygen within said cells on illumination thereof.

Said chromophore may be administered topically or systemically to said patient. For example, said chromophore may be administered by injection.

In accordance with yet another aspect of the invention, there is provided a pharmaceutical composition for administration to a patient for the treatment of a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), which composition comprises a chromophore in accordance with the present invention that is adapted to be delivered to said diseased or undesired cells, and a suitable carrier.

Yet another aspect of the invention envisages a chromophore in accordance with the invention for use in the production of a medicament, for use in the treatment of patients suffering from a disease or disorder which is characterised by the presence in the body of diseased or undesirable cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD); said chromophore being adapted for delivery to said diseased or undesired cells.

Detailed Description of Examples of the Invention

Following are descriptions and examples, by way of illustration only, of embodiments of the invention and methods for putting the invention into effect.

Synthesis of Chromophores Instrumentation and materials

Melting points are uncorrected. Η/¹³C NMR spectra were recorded on Jeol JNM EX270 FT-NMR spectrometer, and are referenced to tetramethylsilane unless otherwise stated. I.R. spectra were obtained using a series 1600 FT-I.R and nominal mass spectra were obtained by Kratos Kompact MALDI II spectrometer. Accurate mass were obtained from EPSRC Mass Spectrometry Service, Swansea. The electronic spectra were obtained using Unicam UV-2 or UV-4 spectrometers and were taken in DCM unless otherwise stated. All reagents and solvents were commercially available and of reagent grade or higher, and were, unless otherwise specified, used as received. TLC analysis were performed on Merck silica-gel 60 plates (F254, 500 μm thickness). Merck Silica-Gel 60 (230-400 mesh) was used for flash chromatographic purification.

Examples 5-(4-isothiocyanatophenyl)-15-(4-pyridiniumyI) porphyrins, dihydroxy chlorins and tetrahydroxybacteriochlorins - General Procedure.

The DPP was synthesised according to the method of Dolphin et al. (1998 5- Phenyldipyrromethane and 5, 15-Diphenylpoφhyrin Org. Synth. 76, 287-293) The resulting mixture of three poφhyrins was chromatographed, eluting initially with DCM to allow removal of 5,15-(4-pyridyl)-DPP, and then continuing with ethyl acetate/DCM (1:4) to elute the required product as puφle crystals. 5-(4-Acetamido phenyl)- 15-(4- methoxyphenyl)poφhyrin (1 eq., 100 mg, 0.182 mmol) was dissolved in 5 M aqueous HC1 (100 mL) and the solution heated for 3 h under reflux. The hot reaction mixture was concentrated in vacuo to yield a crude green solid. The solid was re-dissolved in a mixture of DCM/triethylamine (9:1) (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL), saturated brine (200 L) and the organic layer separated and dried (Na₂S0 ), then concentrated in vacuo. The crude puφle solid was chromatographed, eluting with DCM, and gave 5-(4- Aminophenyl)-15-(4-methoxyphenyl)poφhyrin, as a puφle crystalline solid. The amino group was protected as follows: To a stirred solution of 5-(4-Aminophenyl)-15-(4- methoxyphenyl)poφhyrin, (28 mg, 55 μmol) in anhydrous 1,4-dioxane (2.5 mL) was added solid sodium hydrogen carbonate (6 eq., 28 mg, 0.33 mmol). To this mixture was then added a solution of 9-fluorenylmethyl chloroformate (2 eq., 0.11 mmol, 28.5 mg) in 1,4-dioxane (0.5 mL) under N₂. The reaction flask was covered with aluminium foil to exclude light and stirred at room temperature for a period of 3 h. At this time the reaction was complete (as monitored by TLC). The 1,4-dioxane was removed in-vacuo and the residue partitioned between water (25 mL) and DCM (2 x 25 mL). The combined organic extracts were washed with saturated brine (25 mL) then dried (Na₂SO₄), filtered and concentrated in vacuo. The required poφhyrin was obtained by chromatography, eluting with DCM. The desired poφhyrin was obtained as puφle crystals. The resulting (Fmoc) protected poφhyrin was then used as a stock for the preparation of 5-(4-isothiocyanato)- 15-(4-pyridiniumyl) poφhyrins, 5-(4-isothiocyanato)-15-(4-pyridiniumyl) dihydroxychlorins and 5-(4-isothiocyanato)-15-(4-pyridiniumyl) tetrahydroxybacteriochlorins as follows: 5-(4-isothiocyanato)-15-(4-pyridiniumyl) poφhyrin (35 mg, 48.0 μmol) was converted into the required mixture of chlorin or bacteriochlorin stereoisomers by minor modification of the procedure of Sutton et al. (2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging J. Porphyrin and Phthalocyanines 4, 655-658) using 1 or 2 equivalents of OsO as required. Stocks of 5-(4-N-Fmoc aminophenyl)-15-(4-pyridyl) poφhyrins, 5-(4-N-Fmoc aminophenyl)-15- (4-pyridyl) dihydroxychlorin or 5-(4-N-Fmoc aminophenyl)-15-(4-pyridyl) tetrahydroxybacteriochlorin were deprotected with piperidine (5 equivalents) in DCM and converted to the respective isothiocyanates using the following general procedure: To a stirred solution of 5-(4-aminophenyl) DPP, DP chlorin or DP bacteriochlorin (100 mg, 0.137 mmol) in freshly distilled THF (25 mL) was added l,l'-thiocarbonyldi-2(lH pyridone (64 mg, 0.276 mmol). The reaction was allowed to proceed under argon for 4 hours at room temperature. Excess solvent was evaporated in vacuo to yield a crude puφle solid. The solid was dissolved in a minimal amount of chloroform/methanol (9:1) and purified by flash chromatography (silica). Finally stocks of 5-(4- isothiocyanatophenyl)-15-(4-pyridyl) poφhyrin, chlorin or bacteriochlorin, respectively, were split into batches and reacted independently with 2-bromoethanoic acid, 2- bromoethanol, 2-bromoethanesulphonic acid, 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-mannopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α- galactopyranosyl bromide or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose bromide according to the following procedure: To a solution of the 15-pyridyl poφhyrin, chlorin or bacteriochlorin (50 mg, 0.074 mmol) in anhydrous DMF (10 mL, distilled from CaΗ₂, 0.1 torr) was added the bromide or iodide ( 0.016 mol). The reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1: 1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr) at 30-40 °C to yield the above compound as a lustrous puφle solid. The pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4:1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane. 5-(4-isothiocyanatophenyl)-15-(4-methyIphosphonium) porphyrins, dihydroxy- chlorins and tetrahydroxy-bacteriochlorins - General Synthetic Procedure 1

Boc N-protected 5-(4-aminophenyl)-15-(4-carbomethoxyphenyl) poφhyrin was synthesised by 2+2 condensation methodology via the appropriately substituted 5- phenyldipyrromethanes as described by Boyle et al (Boyle, R.W., Bruckner, C, Posakony, J., James, B.R., Dolphin, D. (1999) Organic Syntheses. 76, 287). The (4- carbomethoxvphenyl) group was converted to a (4-(l-bromomethyl)phenyl) group using the following standard procedure: the poφhyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HC1 (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml). The organic layer was dried (MgS0 ) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted poφhyrin. The stock poφhyrin was then used to generate the corresponding dihydroxychlorin or tetrahydroxybacteriochlorin by treatment with Os0 following the procedure of Sutton et al.(2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging I. Porphyrin and Phthalocyanines 4, 655-658). (4-(l-Hydroxymethyl)phenyl) substituted poφhyrin, dihydroxychlorin or tetrahydroxybacteriochlorin (0.2 mmol) was dissolved in dry chloroform (40 ml) and stirred under argon while triphenylphosphine(1.0 mmol) and carbon tetrabromide (1.6 mmol) were added. The reaction was stirred, in the dark, for 24 hours and then monitored by TLC. Once the hydroxymethyl group had been converted to a bromomethyl group the reaction mixture was diluted with dichloromethane (40 ml), washed with saturated sodium bicarbonate (2 x 20 ml) then brine (2 x 20 ml) and the organic layer dried (MgS0 ). Removal of solvent by evaporation in vacuo afforded the corresponding 4-(l-bromomethylphenyl) poφhyrin, chlorin or bacteriochlorin as puφle crystalline solids. Boc N-protected 5-(aminophenyl)-15-(4-(l-bromomethylphenyl) poφhyrin, chlorin or bacteriochlorin (0.75 mmol) were dissolved in dry dichloromethane (50 ml) under an atmosphere of argon at 25°C. Triethylphosphine, tri-(sulphonatophenyl)phosphine, tri- (carboxyphenyl)phosphine or tri-(hydroxyphenyl)phosphine (7.5 mmol) dissolved in dry dichloromethane (10 ml) were injected by syringe and the progress of the reaction was followed by TLC. Upon completion the solvent was evaporated from the reaction in vacuo and the crude product was purified by flash column chromatography (silica; gradient elution: dichloromethane to methanol) to give the required Boc-N-protected-5- (aminophenyl)-15-(4-methylphosphonium) poφhyrins, chlorins or bacteriochlorins as lustrous puφle crystalline solids. The Boc protecting group was removed by dissolution of the poφhyrin, chlorin or bacteriochlorin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (silica; gradient: dichloromethane to methanol) gave the 5-(4-aminophenyl)-15-(4-methylphosphonium) poφhyrins, chlorins and bacteriochlorins which were converted to the required mono-4- (isothiocyanatophenyl) compounds by treatment with l, -thiocarbonyldi-2(lH)-pyridone using standard procedures (Clarke, OJ. and Boyle, R.W. (1999) .C.S. Chem. Commun. 2231).

5-(4-Isothiocyanatophenyl)-10,15,20-tri(4-methylphosphoniumphenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-(4-methylphosphoniumphenyl)- porphyrin - General Synthetic Procedure 2

Fmoc N-protected 5-(4-aminophenyl)-10,15,20-tri-(4-carbomethoxyphenyl) poφhyrin and 5-(4-aminophenyl)-15-(4-carbomethoxyphenyl) poφhyrin were synthesised by mixed condensation using Lindsey conditions,¹ or by 2+2 condensation methodology via the appropriately substituted 5-phenyldipyrromethanes as described by Boyle et al,² respectively. The (4-carbomethoxyphenyl) groups on these poφhyrins were then converted to (4-(l-bromomethyl)phenyl) groups using the following standard procedure: the poφhyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HC1 (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml). The organic layer was dried (MgS0 ) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted poφhyrins, bearing three or one reduced carbomethoxy groups respectively. 4-(l-Hydroxymethyl)phenyl) substituted poφhyrins (0.2 mmol) were dissolved in dry 1,4-dioxane (40 ml) and stirred under argon while phosphorous tribromide(1.6 mmol) was added. The reaction was stirred, in the dark, for 24 hours and then monitored by TLC. Once all the hydroxymethyl groups had been converted to bromomethyl groups the reaction mixture was diluted with dichloromethane (40 ml), washed with saturated sodium bicarbonate (2 x 20 ml) then brine (2 x 20 ml) and the organic layer dried (MgSO ). Removal of solvent by evaporation in vacuo, followed by column chromatography (silica; CH₂Cl₂.T-.tOAc (9: 1)) afforded the corresponding bromomethyl poφhyrins as puφle crystalline solids.

Fmoc N-protected 5-(aminophenyl)-10,15,20-tri-(4-bromomethylphenyl) poφhyrin and 5-(aminophenyl)-15-(4-bromomethylphenyl) poφhyrin (0.75 mmol) were dissolved in dry DMF (50 ml) under an atmosphere of argon at 25°C. Triaryl or trialkylphosphine (7.5 mmol) was added and the progress of the reaction was followed by TLC. Upon completion the solvent was evaporated from the reaction in vacuo and the crude product was redissolved in a minimal amount of methanol and precipitated by addition of diethyl ether to give the required Fmoc-N-protected-5-(aminophenyl)-methylphosphonium- meso-aryl poφhyrins as lustrous puφle crystalline solids. The Fmoc protecting group was removed by dissolution of the poφhyrin in methanol or acetonitrile, depending upon solubility, and addition of piperidine (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (Cj₈ silica; gradient: water (0.1% TFA)to methanol) gave the 5-(aminophenyl)-methylphosphonium-meso-aryl poφhyrins which were converted to the required mono-4-(isothiocyanatophenyl) compounds by treatment with l, -thiocarbonyldi-2(lH)-pyridone using standard procedures.³ 5-(4-Isothiocyanatophenyl)-15-aryl-10,20-(l,2-dihydroxyethyl)-porphyrins, dihydroxychlorins and tetrahydroxybacteriochlorins - General Synthetic Procedure

The Fmoc protected 5-(4-aminophenyl)-15-aryl poφhyrin (0.8 mmol) was dissolved in dry chloroform (300 ml) under an atmosphere of argon. Freshly recrystallised N- bromosuccinimide (1.8 mmol) in dry chloroform (20 ml) was injected by syringe and the mixture was stirred for 30 min. The solvent was then evaporated in vacuo and the crude product purified by flash column chromatography (silica; gradient elution: hexane to ethyl acetate) to give the required 5, 15-dibromo-10, 20-diarylpoφhyrin as a puφle crystalline solid. The product was then metallated by refluxing in a chloroform/methanol (9: 1) solution of zinc acetate dihydrate (80 mmol). The metallation was followed by visible spectroscopy and, upon completion, was passed through a short column of neutral alumina to remove uncoordinated zinc. The zinc 5,15-dibromo-10, 20-diarylpoφhyrin (0,6 mmol) was dissolved in dry THF to which had been added tetrakis(triphenylphosphine)-palladium(0) (0.6 mmol) and vinyltributyltin (1.4 mmol). The mixture was refluxed under nitrogen for 48 hours after which the solvent was evaporated in vacuo and the residue chromatographed by flash column (silica; gradient elution: dichloromethane to ethyl acetate) to give zinc 5-(Fmoc aminophenyl)-15-aryl- 10,20-diethenyl poφhyrin as a puφle crystalline solid. Zinc 5-(Fmoc aminophenyl)-15- aryl-10,20-diethenyl poφhyrin was demetallated by dissolution in a solution of trifluoroacetic acid in dichloromethane (1% v/v) to give 5-(Fmoc aminophenyl)-15-aryl- 10,20-diethenyl poφhyrin after extracting with water and evaporation of solvent from the organic layer in vacuo. Finally the 10 and 20 ethenyl groups were hydroxylated by osmium tetroxide as described (Sutton J, Fernandez N, Boyle RW (2000) J. Poφhyrins and Phthalocyanines 4, 655), however due to the rapidity of the reaction between the ethenyl groups and osmium tetroxide it was possible to selectively hydroxylate these groups by control of reaction time and stoichiometry. In a typical set of conditions the 5- (Fmoc aminophenyl)-15-aryl-10,20-diethenyl poφhyrin, when treated with osmium tetroxide (5 equivalents) in 10% pyridine/chloroform for 24 - 48 hours, gave the desired 5-(Fmoc aminophenyl)-15-aryl-10,20-bis(l,2-dihydroxyethyl) poφhyrin, while if longer reaction times (72 hours) and higher molar ratios of osmium tetroxide (7.5 or 10 equivalents) are used under the same conditions 5-(Fmoc aminophenyl)-15-aryl-10,20- bis(l,2-dihydroxyethyl) 7,8-dihydroxychlorin and 5-(Fmoc aminophenyl)- 15-aryl- 10,20- bis(l,2-dihydroxyethyl) 7,8,17,18-tetrahydroxybacteriochlorin respectively are obtained. All the above products are converted cleanly to the corresponding isothiocyanates upon piperidine mediated deprotection of the amino group (see above) and treatment with 1,1'- thiocarbonyldi-2(lH)-pyridone using standard procedures (Clarke, O.J. and Boyle, R.W. (1999) .C.S. Chem. Commun. 2231). The 15-aryl group can be substituted either before or post hydro cylations depending upon the nature of the substituent to be introduced and example of this is given for a 15-(4-pyridyl) group which can be quarternised post hydroxylations as follows: Stocks of 5-(4-isothiocyanatophenyl)-10,20-(l,2 dihydroxyethyl)-15-(4-pyridyl) poφhyrin, chlorin or bacteriochlorin, respectively, were split into batches and reacted independently with methyl iodide, 2-bromoethanoic acid, 2- bromoethanol, 2-bromosulphonic acid, 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-mannopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α- galactopyranosyl bromide or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose bromide according to the following procedure: To a solution of the 15-pyridyl poφhyrin, chlorin or bacteriochlorin (50 mg, 0.074 mmol) in anhydrous DMF (10 mL, distilled from CaH₂, 0.1 torr) was added the bromide or iodide (0.016 mol). The reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1:1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr) at 30-40 °C to yield the above compound as a lustrous puφle solid. The pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4:1). Sodium methanolate in dry methanol (1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane.

5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphono-di-ethoxy)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphono-di- ethoxy)phenyl)- porphyrin - General Synthetic Procedure 1 Boc N-protected 5-(4-amιnophenyl)-10,l5,20-tn-(4-bromomethylphenyl) poφhyπn and 5-(amιnophenyl)-15-(4-bromomethylphenyl) poφhyπn (0 75 mmol) were dissolved in a mixture of tπethyl phosphite (15 mmol) and dry acetonitπle (50 ml). A reflux condenser was fitted and the reaction was refluxed under argon. The reaction was followed by TLC and upon completion was washed with saturated sodium bicarbonate (2 x 20 ml), water (2 x 20 ml) and bπne (2 x 20 ml) The organic layer was then dπed (MgS0 ) and the solvent evaporated in vacuo The crude product was then puπfied by flash column chromatography (silica, gradient elution: dichloromethane to ethyl acetate) to give the title compounds as puφle crystalline solids The methylphosphono-di-ethoxy groups were then deprotected to methylphosphono-mono-ethoxy sodium groups by somcation in aqueous sodium hydroxide for 1 hour followed by reversed phase medium pressure chromatography (Cι , gradient elution 0 1% aqueous TFA to methanol) (Boyle, R.W. and van Lier, J E (1993) Synlett 351), or to the fully deprotected methylphosphonic acids by treatment with bromotπmethylsilane (2 equivalents per methylphosphono-di-ethoxy group) for 2 hours followed by reversed phase chromatographic purification chromatography (Cι₈, gradient elution 0 1% aqueous TFA to methanol) Boc deprotection (see above) followed by conversion of the unmasked 4-(amιnophenyl) group to its isothiocyanato analogue was performed using standard procedures (Clarke, O.J and Boyle, R W (1999) 7 C S Chem Commun. 2231)

5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphono-di-ethoxy)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphono-di- ethoxy)phenyl)- porphyrin - General Synthetic Procedure 2

Fmoc N-protected 5-(4-amιnophenyl)-10,15,20-tπ-(4-bromomethylphenyl) poφhyπn and 5-(anrunophenyl)-15-(4-bromomethylphenyl) poφhyπn (0.75 mmol) were dissolved in a mixture of tπethyl phosphite (15 mmol) and dry acetonitπle (50 ml). A reflux condenser was fitted and the reaction was refluxed under argon. The reaction was followed by TLC and upon competion was wahed with saturated sodium bicarbonate (2 x 20 ml), water (2 x 20 ml) and bπne (2 x 20 ml) The organic layer was then dried (MgSO ) and the solvent evaporated in vacuo. The crude product was then purified by flash column chromatography (silica; gradient elution: dichloromethane to ethyl acetate) to give the title compounds as puφle crystalline solids. The methylphosphono-di-ethoxy groups were then deprotected to either methylphosphono-mono-ethoxy sodium groups by sonication in aqueous sodium hydroxide for 1 hour followed by reversed phase medium pressure chromatography (Cι₈; gradient elution 0.1% aqueous TFA to methanol),⁴ or to the fully deprotected methylphosphonic acids by treatment with bromotrimethylsilane (2 equivalents per methylphosphono-di-ethoxy group) for 2 hours followed by reversed phase chromatographic purification chromatography (Cι₈; gradient elution 0.1% aqueous TFA to methanol).⁵ Fmoc deprotection (see above) followed by conversion of the unmasked 4-(aminophenyl) group to it's isothiocyanato analogue was performed using standard procedures.³

5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphonato-di-ethoxy)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphonato-di- ethoxy)phenyl)- porphyrin - General Synthetic Procedure

Boc N-protected 5-(aminophenyl)-10,15,20-tri-(4-hydroxymethylphenyl) poφhyrin and 5-(aminophenyl)-15-(4-hydroxymethylphenyl) poφhyrin (0.75 mmol) were dissolved in a mixture of dry dichloromethane and pyridine (4: 1) under an atmosphere of argon. Diethyl chlorophosphate (2 equivalents per hydroxymethyl group) was injected and the mixture was stirred for 16 hours. Evaporation of solvent from the reaction mixture followed by chromatographic purification gave the corresponding tri or mono ((4- methylphosphonato-di-ethoxy)phenyl) poφhyrins. Treatment with aqueous sodium hydroxide (1M) gave the sodium salts of tri or mono ((4- methylphosphonatoethoxy)phenyl) poφhyrins (Boyle, R.W. and van Lier, J.E. (1995) Synthesis 1079). Boc deprotection and generation of the isothiocyanato group were performed as follows: The Boc protecting group was removed by dissolution of the poφhyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (silica; gradient: dichloromethane to methanol) gave the corresponding 4-(aminophenyl) poφhyrin in quantitative yield.

5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylpyridiniumyl)phenyl)- porphyrin and 5-(4-isothiocyanatophenyI)-15-((4-methylpyridiniumyl)phenyl)- porphyrin - General Synthetic Procedure 1

Boc N-protected 5-(aminophenyl)-10,15,20-tri-(4-bromomethylphenyl) poφhyrin and 5- (aminoophenyl)-15-(4-bromomethylphenyl) poφhyrin (0.75 mmol) were dissolved separately in dichloromethane (50 ml) and 3,5-dicarboxypyridine, 4-(2- sulphonatoethyl)pyridine or 2,6-di-(methylhydroxy)pyridine (15 mmol), as required, were added. A reflux condenser was fitted and the reaction was refluxed under argon. The reaction was followed by TLC and, upon completion, was evaporated to dryness in vacuo. The residue was purified by reversed phase medium pressure chromatography ( ₈; gradient elution 0.1% aqueous TFA to methanol) to yield the N-Boc protected 4- aminophenyl compounds. Deprotection of the aminophenyl group(s) and conversion to the isothiocyanato analogue(s) were conducted as follows: The Boc protecting group was removed by dissolution of the poφhyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (silica; gradient: dichloromethane to methanol) gave the corresponding 4-(aminophenyl) poφhyrin in quantitative yield.

5-(4-Isothiocyanatophenyl)-I0,15,20-tri((4-methylpyridiniumyl)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylpyridiniumyl)phenyl)- porphyrin - General Synthetic Procedure 2 Fmoc N-protected 5-(aminophenyl)-10,25,20-tri-(4-bromomethylphenyl) poφhyrin and 5-(aminoophenyl)-15-(4-bromomethylphenyl) poφhyrin (0.75 mmol) were dissolved in dichloromethane (50 ml) and pyridine (15 mmol), or substituted pyridine (15 mmol), as required, were added. A reflux condenser was fitted and the reaction was refluxed under argon. The reaction was followed by TLC and, upon completion, was evaporated to dryness in vacuo. The residue was purified by reversed phase medium pressure chromatography (Cι₈; gradient elution 0.1% aqueous TFA to methanol) to yield the N- Fmoc protected 4-aminophenyl compounds. Deprotection of the aminophenyl group(s) and conversion to the isothiocyanato analogue(s) were conducted using the standard protocols (see above).

5-(4-Isothiocyanatophenyl)-15-aryl-10,20-(l-hydroxyethyl)-porphyrins, dihydroxychlorins and tetrahydroxybacteriochlorins - General Synthetic Procedure

The Fmoc protected 5-(4-aminophenyl)- 15-aryl poφhyrin (0.8 mmol) was dissolved in dry chloroform (300 ml) under an atmosphere of argon. Freshly recrystallised N- bromosuccinimide (1.8 mmol) in dry chloroform (20 ml) was injected by syringe and the mixture was stirred for 30 min. The solvent was then evaporated in vacuo and the crude product purified by flash column chromatography (silica; gradient elution: hexane to ethyl acetate) to give the required 5, 15-dibromo-10, 20-diarylpoφhyrin as a puφle crystalline solid. The product was then metallated by refluxing in a chloroform/methanol (9:1) solution of zinc acetate dihydrate (80 mmol). The metallation was followed by visible spectroscopy and, upon completion, was passed through a short column of neutral alumina to remove uncoordinated zinc. The zinc 5,15-dibromo-10, 20-diarylpoφhyrin (0,6 mmol) was dissolved in dry THF to which had been added tetrakis(triphenylphosphine)-palladium(0) (0.6 mmol) and vinyltributyltin (1.4 mmol). The mixture was refluxed under nitrogen for 48 hours after which the solvent was evaporated in vacuo and the residue chromatographed by flash column (silica; gradient elution: dichloromethane to ethyl acetate) to give zinc 5-(Fmoc aminophenyl)- 15-aryl - 10,20-diethenyl poφhyrin as a puφle crystalline solid. Zinc 5-(Fmoc aminophenyl)- 15- aryl- 10,20-diethenyl poφhyrin was demetallated by dissolution in a solution of tπfluoroacetic acid in dichloromethane (1% v/v) to give 5-(Fmoc aminophenyl)- 15-aryl- 10,20-dιethenyl poφhyπn after extracting with water and evaporation of solvent from the organic layer in vacuo Finally the 10 and 20 ethenyl groups were epoxidised by treatment with meta-perchlorobenzoic acid (MCPBA) (3 equivalents) and then πng opened by treatment of the bιs-(10,20-epoxyethane) poφhyπn with polyethylene glycol, 2,3,4,6-tetra-O-acetyl-α-glucopyranose, 2,3,4,6-tetra-0-acetyl-α-mannopyranose, 2,3,4,6-tetra-O-acetyl-α-galactopyranose, 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose, orthogonally protected lysine, cysteine, tyrosine, aspartic acid, glutamic acid, seπne, threonιne 2,3,4,6-tetra-0-acetyl-α-glucopyranosyl-l-thιol, 2,3,4,6-tetra-O-acetyl-α- mannopyranosyl-1-thιol, 2,3,4,6-tetra-O-acetyl-α-galactopyranosyl-l-thιol or 2,3,4,6- 2',3',4',6' octa-O-acetyl-α-maltose- 1-thιol (5 equivalents). The resulting 10,20-bιs-(l- hydroxyethyl) poφhyπns were converted into the corresponding dihydroxychloπn and tetrahydroxybacteπochloπn by treatment with Os0 following the procedure of Sutton et α/.(2000 Functionahsed diphenylchloπns and bacteπochloπns - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging J Porphyrin and Phthalocyamnes 4, 655-658) Deprotection of 10,20-bιs 1-hydroxy-sugar and 10,20- bis 1-hydroxy-amιno acid substituted poφhyπns, dihydroxychloπns and tetrahydroxybacteπochloπns was accomplished using standard methodologies which are well known in the art The 15-aryl group can be substituted either pre- or post- hydroxylation depending upon the nature of the substituent to be introduced, an example of this is given for a 15-(4-pyπdyl) group which can be quartermsed post hydroxylations as follows. Stocks of 5-(4-ιsothιocyanatophenyl)-10,20-(l,2 dιhydroxyethyl)-15-(4- pyπdyl) poφhyπn, chlorin or bacteπochloπn, respectively, were split into batches and reacted independently with methyl iodide, 2-bromoethanoιc acid, 2-bromoethanol, 2- bromosulpho c acid, 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl bromide, 2,3,4,6-tetra-0- acetyl-α-mannopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-galactopyranosyl bromide or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose bromide according to the following procedure: To a solution of the 15-pyπdyl poφhyπn, chloπn or bacteπochloπn (50 mg, 0.074 mmol) in anhydrous DMF (10 mL, distilled from CaH2, 0.1 torr) was added the bromide or iodide ( 0016 mol). The reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (normal phase silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1 : 1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 ton) at 30-40 °C to yield the above compound as a lustrous puφle solid. The pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol (1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane.

5-(4-Isothiocyanatophenyl)-10,15,20-tri(4-glycosylphenyl)- porphyrin and 5-(4- isothiocyanatophenyl)-15-(4-glycosylphenyl)- porphyrin - General Synthetic Procedure

2,3.4,6-tetra-O-acetyl-α-glucopyranosyl benzaldehyde, 2,3,4,6-tetra-O-acetyl-α- mannopyranosyl benzaldehyde, 2,3,4,6-tetra-O-acetyl-α-galactopyranosyl benzaldehyde or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose benzaldehyde were condensed independently with 4-nitrobenzaldehyde and pyrrole using Lindsey conditions (Sol, V., Blais, J.C., Cane, V., Granet, R., Guilloton, M., Spiro, M, Krausz, P. (1999) I. Org. Chem. 64, 4431). The crude reaction mixture was purified by flash column chromatography to give 5-(4-nitrophenyl)-10,15,20-tris[4-(2',3',4',6'-tetra-O-acetyl-β- glucopyranosyloxy)phenyl] poφhyrin, 5-(4-nitrophenyl)-10,15,20-tris[4-(2,3,4,6-tetra-O- acetyl-α-mannopyranosyl)phenyl] poφhyrin, 5-(4-nitrophenyl)- 10, 15,20-tris[4-(2, 3,4,6- tetra-0-acetyl-a-galactopyranosyl)phenyl] poφhyrin, or 5-(4-nitrophenyl)-10,15,20- tris[4-(2,3,4,6-2',3',4^,,6' octa-0-acetyl-α-maltose)phenyl] poφhyrin. Alternatively, 4- (2',3',4',6'-tetra-0-acetyl-β-D-glucopyranosyloxy)benzaldehyde, 2,3,4,6-tetra-O-acetyl- α-mannopyranosyl benzaldehyde, 2,3,4,6-tetra-O-acetyl-α-galactopyranosyl benzaldehyde or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose benzaldehyde were used to synthesise 5-(4-(2',3',4',6'-tetra-0-acetyl-β-D-glucopyranosyloxy)phenyl) dipyrromethane, , 2,3,4,6-tetra-O-acetyl-α-mannopyranosyl dipyrromethane, 2,3,4,6- tetra-O-acetyl-α-galactopyranosyl dipyrromethane or 2,3,4,6-2',3',4',6' octa-O-acetyl-α- maltose dipyrromethane using the method of Boyle (Boyle, R.W., Bruckner, C, Posakony, J., James, B.R., Dolphin, D. (1999) Organic Syntheses. 76, 287) which was then condensed to give 5-(4-nitrophenyl)-15,-[4-(2',3',4',6'-tetra-0-acetyl-β- glucopyranosyloxy)phenyl] poφhyrin, 5-(4-nitrophenyl)-15,-[4-(2,3,4,6-tetra-O-acetyl- α-mannopyranosyl)phenyl] poφhyrin, 5-(4-nitrophenyl)-15,-[4-(2,3,4,6-tetra-O-acetyl-α- galactopyranosyl)phenyl] poφhyrin or 5-(4-nitrophenyl)-15,-[4-(2,3,4,6-2',3',4',6' octa-O- acetyl-α-maltose)phenyl] poφhyrin, respectively. Reduction of the nitro group of these poφhyrins was performed by dissolution in THF and addition of 10% palladium on carbon. Stirring of the mixture under H₂ for 5 hours followed by filtration through Celite and purification by flash column chromatography gave the corresponding amino poφhyrins, which were N-protected by reaction with Fmoc chloride (2 equivalents) in anhydrous 1,4-dioxane in the presence of sodium bicarbonate (6 equivalents) under argon. The reaction was monitored by TLC and, upon completion, diluted with dichloromethane and washed with water then brine before drying the organic layer (MgS0 ). Purification by flash column chromatography gave the Fmoc N-protected 5-(4- aminophenyl)-10,15,20-tris[4-(O-acetyl-sugar)phenyl] poφhyrins or 5-(4-aminophenyl)- 15,-[4-(0-acetyl-sugar)phenyl] poφhyrin. N and O protecting groups were removed by dissolution of the poφhyrin in dichloromethane/moφholine (1:1) and stirring for 1 hour. Removal of solvent by evaporation in vacuo was followed by redissolution of the residue in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin was recovered by precipitation with hexane. Finally, the 5-(4-aminophenyl) poφhyrin was dissolved in dry methanol and l,l'-thiocarbonyldi- 2(lH)-pyridone (2 equivalents) was added. The reaction was stirred under argon for 2 hours and monitored by TLC, upon completion, solvent was evaporated in vacuo and the crude product was purified by preparative medium pressure reversed phase chromatography (Cι₈;gradient elution: 0.1% aqueous TFA to methanol).

5-(4-isothiocyanatophenyl)-15-(4-α-glucopyranosyl, -α-mannopyranosyl, -α- galactopyranosyl or -α-maltose ) porphyrins, dihydroxy chlorins and tetrahydroxybacteriochlorins - General Procedure. The required DDP was synthesised according to the method of Dolphin et α/.(1998 5- Phenyldipyrromethane and 5, 15-Diphenylpoφhyrin Org. Synth. 76, 287-293) The resulting mixture of three poφhyrins was chromatographed, eluting initially with DCM to allow removal of 5,15-(4-hydroxyphenyl)-DPP, and then continuing with ethyl acetate/DCM (1:4) to elute the required product as puφle crystals. 5-(4-Acetamido phenyl)- 15-(4-methoxyphenyl)poφhyrin (1 eq., 100 mg, 0.182 mmol) was dissolved in 5 M aqueous HC1 (100 mL) and the solution heated for 3 h under reflux. The hot reaction mixture was concentrated in vacuo to yield a crude green solid. The solid was re- dissolved in a mixture of DCM/triethylamine (9: 1) (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL), saturated brine (200 mL) and the organic layer separated and dried (Na2S0 ), then concentrated in vacuo. The crude puφle solid was chromatographed, eluting with DCM, and gave 5-(4- Aminophenyl)-15-(4-methoxyphenyl)poφhyrin, as a puφle crystalline solid. The amino group was protected as follows: To a stirred solution of 5-(4- Aminophenyl)- 15-(4- methoxyphenyl)poφhyrin, (28 mg, 55 μmol) in anhydrous 1,4-dioxane (2.5 mL) was added solid sodium hydrogen carbonate (6 eq., 28 mg, 0.33 mmol). To this mixture was then added a solution of 9-fluorenylmethyl chloroformate (2 eq., 0.11 mmol, 28.5 mg) in 1,4-dioxane (0.5 mL) under N₂. The reaction flask was covered with aluminium foil to exclude light and stirred at room temperature for a period of 3 h. At this time the reaction was complete (as monitored by TLC). The 1,4-dioxane was removed in vacuo and the residue partitioned between water (25 mL) and DCM (2 x 25 mL). The combined organic extracts were washed with saturated brine (25 mL) then dried (Na₂S0 ), filtered and concentrated in vacuo. The required poφhyrin was obtained by chromatography, eluting with DCM. The desired poφhyrin was obtained as puφle crystals. The resulting (Fmoc) protected poφhyrin was then used as a stock for the preparation of 5-(4-isothiocyanato)- 15-(4-α-glucopyranosyl, -α-mannopyranosyl, -α-galactopyranosyl or -α-maltose) poφhyrins, 5-(4-isothiocyanato)-15-(4~α-glucopyranosyl, -α-mannopyranosyl, -α- galactopyranosyl or -α-maltose) dihydroxychlorins and 5-(4-isothiocyanato)-15-(4-α- glucopyranosyl, -α-mannopyranosyl, -α-galactopyranosyl or -α-maltose) tetrahydroxybacteriochlorins as follows: 5-(4-isothiocyanato)-15-(4-hydroxyphenyl) poφhyrin (35 mg, 48.0 μmol) was converted into the required mixture of chlorin or bacteriochlorin stereoisomers by minor modification of the procedure of Sutton et al. (2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging J. Porphyrin and Phthalocyanines 4, 655-658) using 1 or 2 equivalents of Os0₄ as required. Stocks of 5-(4-N-Fmoc aminophenyl)- 15-(4-hydroxyphenyl) poφhyrins, 5-(4-N-Fmoc aminophenyl)- 15-(4-hydroxyphenyl) dihydroxychlorin or 5-(4-N-Fmoc aminophenyl)- 15-(4-hydroxyphenyl) tetrahydroxybacteriochlorin were deprotected with piperidine (5 equivalents) in DCM and converted to the respective isothiocyanates using the following general procedure: To a stirred solution of 5-(4-aminophenyl) DPP, DP chlorin or DP bacteriochlorin (100 mg, 0.137 mmol) in freshly distilled THF (25 mL) was added 1,1'- thiocarbonyldi-2(lH)-pyridone (64 mg, 0.276 mmol). The reaction was allowed to proceed under argon for 4 hours at room temperature. Excess solvent was evaporated in vacuo to yield a crude puφle solid. The solid was dissolved in a minimal amount of chloroform/methanol (9: 1) and purified by flash chromatography (silica). Finally stocks of 5-(4-isothiocyanatophenyl)-15-(4-phenol) poφhyrin, chlorin or bacteriochlorin, respectively, were split into batches and reacted independently with 2,3,4,6-tetra-O- acetyl-α-glucopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-mannopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-galactopyranosyl bromide or 2,3,4,6-2',3',4',6' octa-O-acetyl-α- maltose bromide according to the following procedure: To a solution of the 15-phenol poφhyrin, chlorin or bacteriochlorin (50 mg, 0.074 mmol) in anhydrous DMF (10 mL, distilled from CaΗ₂, 0.1 ton) was added the required bromo sugar ( 0.016 mol) and potassium carbonate (0.16 mol). The reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (normal phase silica). Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr) at 30-40 °C to yield the above compound as a lustrous puφle solid. The resulting sugar substituted poφhyrins were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane. 5-(4-isothiocyanatophenyI)-15-(4-methyl-α-S-glucopyranosyI, -α-S - mannopyranosyl, -α-S -galactopyranosyl or -α-S -maltose) porphyrins, dihydroxychlorins and tetrahydroxy-bacteriochlorins - General Synthetic Procedure

Boc N-protected 5-(4-aminophenyl)-15-(4-carbomethoxyphenyl) poφhyrin was synthesised by 2+2 condensation methodology via the appropriately substituted 5- phenyldipyrromethanes as described by Boyle et al. (Boyle, R.W., Bruckner, C, Posakony, J., James, B.R., Dolphin, D. (1999) Organic Syntheses. 76, 287). The (4- carbomethoxyphenyl) group was converted to a (4-(l-bromomethyl)phenyl) group using the following standard procedure: the poφhyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HCl (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml). The organic layer was dried (MgS0₄) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted poφhyrin. The stock poφhyrin was then used to generate the corresponding dihydroxychlorin or tetrahydroxybacteriochlorin by treatment with OsO following the procedure of Sutton et α/.(2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging I. Porphyrin and Phthalocyanines 4, 655-658). (4-(l-Hydroxymethyl)phenyl) substituted poφhyrin, dihydroxychlorin or tetrahydroxybacteriochlorin (0.2 mmol) was dissolved in dry chloroform (40 ml) and stirred under argon while triphenylphosphine( 1.0 mmol) and carbon tetrabromide (1.6 mmol) were added. The reaction was stirred, in the dark, for 24 hours and then monitored by TLC. Once the hydroxymethyl group had been converted to a bromomethyl group the reaction mixture was diluted with dichloromethane (40 ml), washed with saturated sodium bicarbonate (2 x 20 ml) then brine (2 x 20 ml) and the organic layer dried (MgS0₄). Removal of solvent by evaporation in vacuo afforded the corresponding bromomethyl poφhyrin, chlorin or bacteriochlorin as puφle crystalline solids. Boc N-protected 5-(aminophenyl)-15-(4-bromomethylphenyl) poφhyrin, chlorin or bacteriochlorin (0.75 mmol) were dissolved in dry dichloromethane (50 ml) under an atmosphere of argon at 25°C. and 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl- 1-thiol, 2,3,4,6-tetra-O-acetyl-α-mannopyranosyl-l-thiol, 2,3,4,6-tetra-O-acetyl-α- galactopyranosyl- 1-thiol or 2,3,4,6-2',3',4',6' octa-O-acetyl-α-maltose- 1-thiol (7.5 mmol) dissolved in dry dichloromethane (10 ml) were injected by syringe and the progress of the reaction was followed by TLC. Upon completion the solvent was evaporated from the reaction in vacuo and the crude product was purified by flash column chromatography (silica; gradient elution: dichloromethane to methanol) to give the required Boc-N- protected-5-(aminophenyl)-15-(4-methyl-α-2,3,4,6-tetra-0-acetyl-S-glucopyranosyl, - α-2,3,4,6-tetra-0-acetyl -S -mannopyranosyl, -α2,3,4,6-tetra-0-acetyl -S - galactopyranosyl or -α or 2,3,4,6-2',3',4',6' octa -S -maltose) poφhyrins, chlorins or bacteriochlorins as lustrous puφle crystalline solids. The resulting sugar substituted poφhyrins were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane. Finally the Boc protecting group was removed by dissolution of the poφhyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (Cι₈ silica; gradient: dichloromethane to methanol) gave the corresponding 4-(aminophenyl) poφhyrins.

Removal of solvent by evaporation, followed by purification by flash column chromatography (silica; gradient: dichloromethane to methanol) gave the 5-(4- aminophenyl)-15-(4-methyl-α-S-glucopyranosyl, -α-S -mannopyranosyl, -α-S - galactopyranosyl or -α- or -S -maltose) poφhyrins, chlorins and bacteriochlorins which were converted to the required mono-4-(isothiocyanatophenyl) compounds by treatment with l,l'-thiocarbonyldi-2(lH)-pyridone using standard procedures (Clarke, O.J. and Boyle, R.W. (1999) /.C.S. Chem. Commun. 2231)

5-(4-isothiocyanatophenyl)-10,15,20-tris[4-(β-D-gIucopyranosylthio)-2,3,5,6- tetrafluorophenyl)]porphyrin and 5-(4-isothiocyanatophenyl)-15-[4-(β-D- glucopyranosylthio)-2,3,5,6-tetrafluorophenyl)]porphyrin, dihydroxychlorin and tetrahydroxybacteriochlorin - General Synthetic Procedure

Pentafluorobenzaldehyde was condensed with 4-nitrobenzaldehyde and pyrrole using Lindsey conditions or Alder Longo procedure and the crude reaction mixture purified by column chromatography to give 5-(4-nitrophenyl)-10,15,20-tri(pentafluorophenyl)- poφhyrin. Alternatively, using the method of Boyle, 5-(4-nitrophenyl)dipyrromethane and 5-(pentafluorophenyl)dipyrromethane were condensed to give 5-(4-nitrophenyl)-15-( pentafluorophenyl)-poφhyrin. The selective nucleophilic substitutions of the p-¥ atoms in the unsyminetric tetraphenyl and diphenyl poφhyrins were achieved using 2',3',4',6'- tetra-O-acetyl-β-D-thioglucopyranose in DMF at room temperature to give 5-(4- nitrophenyl)-10,15.20-tris[4-(2',3',4',6'-tetra-O-acetyl-β-D-glucopyranosylthio)-2,3,5,6- tetrafluorophenyl)]poφhyrin and 5-(4-nitrophenyl)-15-[4-((2',3',4',6'-tetra-O-acetyl-β- D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl)]poφhyrin. The crude reaction mixture was purified by flash column chromatography. Reduction of the nitro group of these poφhyrins was performed by dissolution in THF and addition of 10% palladium on carbon. Stirring of the mixture under H₂ for 5 hours followed by filtration through Celite and purification by flash column chromatography gave the corresponding amino poφhyrins, which were N-protected by reaction with Fmoc chloride (2 equivalents) in anhydrous 1,4-dioxane in the presence of sodium bicarbonate (6 equivalents) under argon. The reaction was monitored by TLC and, upon completion, diluted with dichloromethane and washed with water then brine before drying the organic layer (MgS0 ). Purification by flash column chromatography gave the Fmoc N-protected 5-(4- aminophenyl)- 10, 15 ,20-tris[4-(2' ,3 ' ,4' ,6' -tetra-Oacetyl-β-D-glucopyranosylthio)- 2,3,5,6-tetrafluorophenyl)]pθφhyrin or 5-(4-aminophenyl)-15-[4-((2',3',4',6'-tetra-0 acetyl-β-D-glucopyranosylthio)-2,3,5,6-tetraflurophenyl)]poφhyrin. At this point the core poφhyrin was either deprotected (see below) or converted to the corresponding di hydroxychlorin or tetrahydroxybacteriochlorin by treatment with Os0₄ followed by reduction of the intermediate osmate ester with H₂S, as described using the standard procedure described for other 5,15-diphenylpoφhyrins. N and O protecting groups were removed by dissolution of the poφhyrin in dichloromethane/moφholine (1/1) and stirring for 1 hour. Removal the solvent by evaporation in vacuo was followed by redissolution of the residue in a mixture of dichloromethane and methanol (4/1). Sodium methanolate in dry methanol (1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected poφhyrin was recovered by precipitation with hexane. Finally, the 5-(4-aminophenyl)poφhyrins were dissolved in dry THF and 1,1 '- thiocarbonyldi-2(lH)-pyridone (2 equivalents) was added. The reaction was stirred under argon for 2 hours and monitored by TLC, upon completion, solvent was evaporated in vacuo and the crude product was purified by preparative medium pressure reversed phase chromatography ( ₈ ; gradient elution : 0,1% aqueous TFA to methanol).

Methodology Description 1 : General Bioconjugation protocol Porphyrin Isothiocyanate (PITC) + Antibody

A stock solution of a PITC in accordance with the invention in DMSO was prepared to a molarity of 0.027, this solution was desiccated and stored at 0°C until required. A solution of antibody was extensively dialysed against sterilised PBS to remove any trace of azide. The dialysed antibody solution was then adjusted to a concentration of 10 mg/mL via centrifugal concentration and separated into 250 μL aliquots.

A I M solution of sodium bicarbonate was prepared and adjusted to pH 9.0 with 2 M sodium hydroxide.

To a 250 μL aliquot of antibody was added 30 μL of 1 M sodium bicarbonate. A predetermined volume of PITC stock solution was then added to give a desired molar ratio (MR) of poφhyrin to antibody. For example an MR of 20 was achieved via the addition of 10 μL of stock solution to 250 μL of antibody at 10 mg/mL. In order to maintain a constant concentration of DMSO in the bioconjugation reaction mixture, all aliquots of stock solution were diluted to 25 μL with further portions of DMSO.

Desired Vol. of [C] of Vol. of 1 M Vol. of Vol. of

MR antibody antibody sodium PITC stock extra solution solution bicarbonate solution DMSO 20 250 μL 10 mg/mL 30 μL 10 μL 15 μL

10 250 μL 10 mg/mL 30 μL 5 μL 20 μL

5 250 μL 10 mg/mL 30 μL 2.5μL 22.5 μL

2.5 250 μL 10 mg/mL 30 μL 1.25 μL 23.75 μL

Table 1.0 Quantities of reagents for bioconjugation

Following addition of PITC the bioconjugation reaction was agitated gently for 1 hour at 25°C. After 1 hour the crude bioconjugation reaction mixture was loaded directly onto the top of a prepacked PD10 size exclusion column pre-equilibrated with sterile PBS (25 mL). The column was eluted with sterile PBS. Antibody-poφhyrin conjugate was eluted in the first coloured band/fraction. The antibody-poφhyrin conjugate concentration following dilution during chromatography was determined as 1.25 mg/mL. The degree of labelling (DOL) of poφhyrin to antibody was calculated via standard spectroscopic methods using known constants of molar absoφtivity for both poφhyrin and protein.

Antibody-poφhyrin conjugates were stored, without further concentration, in PBS + azide at 0°C unless otherwise stated.

Methodology Description 2 : Standard Photocytotoxicity

Cells are grown to confluence or appropriate density then washed 2 times with PBS (phosphate buffered saline) to eliminate all trace of FBS (fcetal bovine serum). Cell density is adjusted to 1.5x106 cells/ml in medium without FBS and these are then incubated for 1 hour in the dark (37 degrees C, 5% C02 ) with a range of photosensitiser/conjugate concentrations. Post incubation, cells are washed further with medium (without FBS )to eliminate unbound photosensitiser, then resuspended and seeded in 96 wells plates (lxlO⁵ cells/well) in quadruplate. Plates are then either irradiated (3.6J/cm2 of filtered red light >600nm ) or left in the dark as "dark toxicity controls" for the same period of time (-14 minutes).Five microliters (5%/well) of FBS is added after the irradiation/dark period and the plates are returned to the incubator overnight. Twenty to 24 hours after treatment, 10 μl of MTT solution (Sigma Thiazolyl blue, 4.8x10^"^ in PBS)is added per well and the plates are returned to the incubator until color develops (between 1 and 4 hours). A solution of acid-alcohol (lOOμl/well of 0.04N HCL in isopropanol) is the added and mixed thoroughly to dissolve the dark blue crystals. Plates are then read at 570nm in a microplate reader and the % cell survival calculated against controls.

Methodology Description 3 ; Initial Flow Cvtometry Chromophore Analysis

The two fluorochromic probes were generated from separate reactions of 2,3- dihydroxy-5-(4-methylphosphoniumtriethyl)-15-(4-isothiocyanatophenyl)chlorin (higher R_f regioisomer) and 2,3, 12,13-tetrahydroxy-5-(4-isothionatophenyl)-15-(4- methylphosphoniumtriethyl) bacteriochlorin (lower R_f cis stereoisomer) with avidin under the standard bioconjugation protocols given earlier. An initial flow experiment has been undertaken utilising these separate avidin conjugates with RAJI cells and biotin monoclonal antibodies (HLA-DRl, L243), (laser excitation 488 nm, collecting emissions at < 640 nm (FL2) > 670 nm (FL3)). Data indicated that the signals from the DPBC samples were much higher due to good match to emission filter (FL3). Samples containing avidin DPCH or DPBC conjugates with L243 antibodies indicated modest increases in fluorescence compared to controls. Using higher concentrations of avidin- DPCH/DPBC the peak fluorescence increased, which may either be due to the initial concentrations of conjugates being too low to saturate receptors or to a lesser extent to some non-covalent binding. Control samples with avidin-DPCH/DPBC (no antibody) showed some background fluorescence in the absence of L243 antibodies, suggesting that some non-specific binding of the conjugates to the RAJI cells had occurred or that a small quantity of non-covalently bound fluorophore had transferred from the protein to the cell surface. A FITC-avidin control indicated that a slightly higher signal was present in FL2 which appears also in FL3 due to a broad emission band. In the presence of L243 antibody the mean signal increased by 150%. This indicates that non-covalent binding is less significant with FITC-avidin conjugates.

Experiments have been undertaken to determine the level of non-covalent binding of fluorophore to the protein surface (BSA and avidin). 'Blank' bioconjugations using mixtures of the unreactive DPCH and DPBC derivatives 2,3-dihydroxy-5-(4- methylphosphoniumtriethyl)-15-(4-acetomidophenyl)chlorin (higher R_f regioisomer) and 2,3,12,13-tetrahydroxy-5-(4-acetomidophenyl)-15-(4- methylphosphoniumtriethyl) bacteriochlorin (higher R_f trans stereoisomer) with both BSA and avidin have been carried out and the resultant protein solutions have been purified by gel filtration (PD-10) as described for the reactive probes described earlier. UV analysis indicated that approximately similar amounts of unreactive probes non-covalently bind to the proteins. For BSA or avidin, 1 unreactive DPCH binds to each protein molecule, whereas DPBC is less than 1 due probably to its increased polarity and non-amphiphilic nature.

Initial studies have been undertaken to remove non-covalently bound fluorophore from the protein (BSA and avidin) using SDS-PAGE. When the 'blank' bioconjugation mixtures were subjected to SDS-PAGE separation of all non-covalently bound fluorophore was achieved (UV/fluorescence of a solubilised gel segment at 66000 D for BSA and 16500 D for avidin monomer indicated no signal). Further to these investigations, we have been able to show that fluorophore which is non-covalently bound to BSA (or avidin) transfers to the surface of HeLa cells. When HeLa cells were added to solutions of the non-covalent fluorophore-protein complexes, and incubated for 20 min, fluorescence was removed from the solution with removal of the HeLa cells. This effect was much more marked with 2,3-dihydroxy-5-(4- methylphosphoniumtriethyl)-15- (4-acetomidophenyl)chlorin (higher R_f regioisomer) than with 2,3,12,13-tetrahydroxy-5- (4-acetomidophenyl)-15-(4- methylphosphoniumtriethyl) bacteriochlorin (higher R_f trans stereoisomer). Re-suspension of the cells and measurement of the fluorescence indicated a 10-fold increase in fluorescence in the case of the DPCH, whereas the DPBC only showed a modest increase. These measurements suggest that there is significant fluorescence quenching of both DPCH and DPBC by the protein and that the DPCH's amphiphilic nature has allowed incoφoration into the HeLa cell membrane resulting in restoration of almost complete fluorescence. The DPBC, being non-amphiphilic, may complex to the surface of the HeLa cell in a similar manner as it does to the protein resulting in similar fluorescence quenching.

Since the fluorophore conjugates can be purified by SDS-PAGE we have investigated the use of preparative electrophoresis as a technique for removal of non- covalently bound fluorophore. To this end we have used a Centrilutor® micro- electroeluter bought from Millipore. This device has allowed recovery of pure protein fluorophore conjugates from SDS gels.

Methodology Description 4 : Elution of Conjugates from SDS PAGE Utilising Micro-Electroeluter

• Working in greatly subdued lighting, the SDS-PAGE of the required protein conjugate was cut into small strips and added to the centrilutor sample tubes and the tops closed (no more than half full, 3-4 sample tube used).

• The lower buffer chamber of the electroeluter was filled with degassed SDS running buffer up to the level of the first electrode.

• 3 to 4 Centricon® centrifugal devices (YM-30 used for BSA conjugates and YM-3 for avidin conjugates) from Millipore were inserted firmly into the holes in the upper buffer chamber rack of the electroeluter from below (with filter membrane lowest) and the vacant holes of the rack were stoppered with stoppers provided, from the underside of the rack.

• The upper buffer chamber was placed into the lower buffer chamber with both electrodes aligned on the same side of the electroelutor.

• The upper buffer chamber was then filled with degassed SDS running buffer (as before) until all Centricon® unit tops were completely immersed. If no leaks were detected the air bubbles trapped below the Centricon® units were removed via an angled plastic pipette(reinforced with paper clip).

• The centrilutor sample tubes were then placed into the top of the Centricon® units, ensuring the sample tube fitted snugly and filled completely with sample buffer (air bubbles were removed as described earlier).

• The safety cover of the electroelutor was added and the power supply connected (200 V, 50 mA used).

• After a period of 2-3 h. the power supply was removed and the Centricon® filter extracted from the upper buffer chamber of the electroelutor.

• The filtrate vial was added to the filter unit and a retentate top added. The excess buffer was then removed by centrifugation at 5000G (BSA) and 7,500G (avidin) for 2 h. Fresh 0.5 M phosphate buffer (pH 7.0) was added to the Centricon® unit and the procedure was repeated to ensure all SDS was removed. • The concentrated purified conjugates were then collected in the retentate vials of the filter units by inversion and centrifugation. Sodium azide (2 M, 20 ml) was added and the conjugates were stored at 4°C.

Methodology Description 5 : FACS Conjugate Binding Protocol

Wash flask of cells with phosphate buffered saline (PBS) pH 7.3. Treat with 5mM EDTA in PBS for 10 min at 37C. Tap flask to dislodge cells, place in 50mL polypropylene tube and pellet at 400g 3 min. Resuspend in 10 mL PBS and count cells. Place 2 x 10⁵ in FACS tube (Falcon 2054) and wash with ImL PBS by centrifugation (400g 3min) and resuspension by agitation.

Block cells in 500 μL 2% Marvel milk powder in PBS, 1% BSA 30 min RT Wash cells in 1 mL PBS/BSA/Azide centifuge and resuspend pellet (as above) Add 10 μL appropriate antibody dilution. Incubate on ice lh Wash cells in 1 mL PBS/BSA/Azide centifuge and resuspend pellet (as above) Add 50 μL Rabbit anti-mouse:FITC (Serotec, 1/100 dilution) and incubate on ice in the dark lh

Wash cells in 1 mL PBS/BSA/Azide centifuge (as above) and resuspend pellet in 400 μL PBS/BSA/Azide.

Run samples through FACS machine using CellQuest acquisition software to collect data.

PBS/BSA/AZIDE

250 mL PBS

0.625g BSA

1.56 mL Sodium Azide (1.6M)

Methodology Description 6 : SDS-PAGE

Separating gel

Component % of gel 5 20

Acrylamide/Bis (40% w/v) 1.67mL 6.66mL 1.5M Tris-HCl (pH 8.8) 2.5mL 2.5mL

Water 5.67mL 0.7mL

TEMED 10 μL 10 μL

10% Ammonium persulphate 50 μL 50 μL

SDS 100 μL 100 μL

For gradient gel 5-20% a gradient mixer connected to a peristaltic pump is used.

Stacking gel (3%)

Component mL

Acrylamide/Bis (40% w/v) 1.3 lM Tris-HCl (pH 6.8) 1.25

Water 7.4

TEMED 20 μL

10% Ammonium persulphate 50 μL

SDS 100 μL

Running buffer

0.025M Tris, 0.192M glycine, 0.1% SDS, pH8.3 in water.

Sample buffer lM Tris-HCl pH 6.8 13mL

20% SDS 6.5mL

Glycerol 5.2mL

0.5% Bromophenol blue 0.26mL

Biorad Protean 2 equipment was used in accordance with manufacturer's instructions

Samples (total volume 15-20 μL containing 1-10 μg sample protein) were loaded onto a gel.

Gels were run at 200V for approximately lh. Gels were then scanned by light, after which they were stained using Coomassie blue stain and subsequently destained using acetic acid/methanol.

Target antibodies and antigens All antibodies are purchased from Serotec Ltd, Kidlington , Oxford, 0X5 1JE

CD 104 (β-4-INTEGRIN) β-4-Integrin is a 205kDa glycoprotein member of the integrin family that associates with α6 integrin to form the α6β4 complex. Integrins are cell surface adhesion molecules that interact with the extracellular matrix and affect cellular moφhology and function. The polarized distribution of integrins is often lost in carcinomas and there have been several reports that α6β4 integrin is upregulated in skin, colorectal and bladder carcinomas. It has also been reported that an increase in α6β4-integrin leads to a more invasive, and hence aggressive, phenotype in colorectal carcinoma.

EpCAM

Epithelial cell adhesion molecule (EpCAM) is a 40kDa cell-cell adhesion molecule. It is expressed at low levels on cells of epithelial origin (colon mucosa and lung acini) but is markedly up-regulated on cancer cells, particularly colorectal and gastrointestinal carcinomas. The role of EpCAM in cancer is unclear but it does have a negative effect on cadherin-mediated adhesion that could promote metastasis. EpCAM is the target of many antibodies, some of which have been used in immunotherapy, these antibodies include 17-1A, MOC-31, 323/A3, GA733, K931, VU1D9, 162-21.2, KS-1/4, AUA-1, MH99, 2G8, 311-1K1 and MM104. 17-1A is the most widely used antibody and has been as adjuvant treatment following surgery to kill residual tumour cells. Phage scFv antibodies have been generated against EpCAM to give better tumour penetration and retention; these antibodies have also been conjugated to give a T-cell/EpCAM bispecific antibody. The GA733 and 323/A3 antibodies have also undergone trials but as they are of higher affinity than 17-1 A they cause more damage to normal cells. The 17-1A antibody has been conjugated to photodynamic molecules and used in PDT by ourselves and others.

CD146 (MUC-18)

MUC-18 (also called Mel-CAM) is a 100-1 lOkDa cell adhesion molecule which is a member of the immunoglobulin gene superfamily. MUC-18 is not expressed in most normal tissues, except in basal cells of bronchial epithelium and endothelial cells. It has been found up-regulated on the surface of numerous tumour types including malignant melanoma and is thought to promote tumour progression, implantation and metastases. Conversely, in some breast cancers low levels of MUC-18 expression were associated with good prognosis. MUC-18 antibodies may have applications in tumour targeting due to the protein core being exposed after loss of the carbohydrate moieties from the molecule during malignancy. A MUC-18 antibody, ABX-MA1, has been shown to inhibit melanoma tumour growth and metastases.

CD4

The CD4 molecule is a single chain transmembrane glycoprotein of 59kDa expressed principally on helper/inducer T cells and monocytes. The CD4 molecule plays a role in the T cell receptor binding of Class II MHC complexes that are found on cells involved in immune regulation, e.g. dendritic cells, B cells and macrophages.

CD8

The CD8 molecule is composed of two glycoprotein chains and has a molecular weight of 32kDa. It is expressed on cytotoxic T cells and NK cells. The CD8 molecule plays a role in the T cell receptor binding of Class I MHC complexes that are found on a large number of human cells.

Further exemplification of the invention

It has been demonstrated in our original work, described inter alia in Sutton, J., Fernandez, N. and Boyle, R.W. (2000) Functionalised Diphenylchlorins and Bacteriochlorins - Their Synthesis and Bioconjugation for Targeted Photodynamic Therapy and Tumour Cell Imaging. I. Porphyrins and Phthalocyanines 4, 655-658; and Clarke, O.J. and Boyle, R.W. (1999) Isothiocyanatopoφhyrins, useful intermediates for the conjugation of poφhyrins with biomolecules and solid supports. .C.S. Chem. Commun. 2231-2232, each of which is incoφorated herein by reference, that a set of poφhyrin, chlorin and bacteriochlorin molecules can be efficiently conjugated to proteins via a stable thiourea bond, and that these conjugates have potential as fluorescence imaging agents.

As exemplification of the present invention, we now describe the use of this method to form conjugates between monoclonal antibodies having high specificity for human cancer cells, and our set of poφhyrin based photosensitisers. Conjugates formed in this way have been assayed for photodynamic activity against the corresponding carcinoma cells, and also for their ability to selectively bind to, and photosensitise, these target cells in the presence of non-target cells. We also demonstrate the specific internalisation of poφhyrin-BSA conjugates into HeLa cells.

Our examples utilise 5-(4-isothiocyanatophenyl)-15-(N- hydroxyethylpyridiniumyl) poφhyrin (HEPP-NCS). A synthetic protocol for this chromophore is described in Example 1.

(HEPP-NCS)

Example 25 - stable conjugation to antibodies

HEPP-NCS was prepared as described in Example 1 above. Antibody 17.1 A was selected for the bioconjugation procedure. 17.1 A is an antibody which reacts specifically with a receptor that is over-expressed on colorectal cancer cells, in particular Colo 320 cells (ECACC, deposit no. 87061205). However, any antibody which reacts against any antigen that is over-expressed on a suitable cell line may be utilised in accordance with the invention. Examples of such antibodies include Ber-EP4 and MOK-31, each of which is commercially available from DAKO Ltd, Ely, Cambridgeshire, and each of which is reactive against an antigen that is over-expressed on epithelial cells.

To increase the buffer pH of the antibody preparation to approximately pH9, prior to and for the puφoses of the bioconjugation procedure, the monoclonal antibody preparation was either buffer-exchanged from a phosphate to an acetate buffer using a Centricon centrifuge or was subjected to dialysis so as to exchange the phosphate buffer for an acetate buffer.

HEPP-NCS was conjugated with 17.1 A monoclonal antibody in accordance with the method described in Methodology Description 1, to obtain a range of conjugation dilutions having respective MRs of 2.5, 5, 10 and 20..

The acetate-buffered antibody preparation and range of conjugation dilutions obtained therefrom were subjected to SDS-PAGE in accordance with the method described in Methodology Description 6.

Neither the buffer-exchange nor dialysis procedures was found to disrupt the antibody structure, the light and heavy chains remaining associated with one another and migrating together on each of the gels.

Example 26 - FACS analysis

FACS analyses were run in accordance with Methodology Description 5.

Example 27 - Photocytotoxicitv experiments

Photocytotoxicity tests in accordance with the method described in Methodology Description 2 were performed on Colo 320 cells utilising various antibody conjugates.

As described in Example 16 OX-34 has been found to lack specificity for any antigens expressed on the surface of Colo 320 cells. Accordingly, as expected these control experiments show no photocytotoxicity following irradiation.

Further control experiments were performed using "capped" HEPP. The "capping" procedure involved reacting the NCS group on HEPP-NCS with propylamine, so as to block serum protein conjugation. No significant cytotoxicity was found in the dark, indicating that HEPP is substantially non-toxic to Colo 320 cells. On irradiation, however, some photocytotoxicity was observed, indicating that an amount of the capped HEPP has been transferred to the surface of the Colo 320 cells.

In the absence of any antibody, transfer of the capped chromophore to the cell membrane is probably attributable to the amphiphilic nature of the capped chromophore, which possesses both hydrophilic groups around the poφhyrin core and a hydrophobic propylamine "capping" group. This renders the chromophore particularly susceptible to becoming embedded in a lipid membrane such as the Colo 320 cell membrane.

A significant increase in cytotoxicity was seen on irradiation, indicating that the binding of the bioconjugate to the cell surface confers photosensitivity upon the cells. Hence, this species is a suitable candidate for PDT.

Example 28 - photodynamic therapy in vivo

Protocols for performing and assessing photodynamic therapy in vivo, utilising the conjugates of the invention, are variously described in R Boyle et al, Br. I. Cancer (1992) 65:813-817; R Boyle et al, Br. I. Cancer (1993) 67:1177-1181; R Boyle et al, Br. I. Cancer (1996) 73:49-53; and Lapointe et al, I. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876-882; the contents of each of which are incoφorated herein by reference.

As described in these papers, tumours may be induced or transplanted into animals such as mice, and the animal may then be injected with a quantity of photosensitiser in accordance with the invention conjugated to an antibody with specificity for an antigen which is specifically expressed or over-expressed on the surface of the tumour cells. Thereafter, the animal may be subjected to irradiation, and the effects on the tumour assessed, qualitatively or metrically, with reference to tumour metabolism (as described in Lapointe et al, I. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876- 882). As described in R Boyle et al, Br. I. Cancer (1996) 73:49-53, the distribution of the photosensitiser in vivo may also be measured, by biodistribution and/or vascular stasis assays.

Example 29 - Confocal Laser Scanning Microscopy

A preliminary examination of the intracellular localisation of a conjugate of 5- isothiocyanatophenyl- 15-(N-hydroxyethylpyridiniumyl)poφhyrin (HEPP-NCS) with BSA was earned out using confocal laser scanning microscopy. The readily available epithelial human carcinoma cell line HeLa was selected for incubation with the conjugate. All incubations were performed in tπphcate with sub-confluent cultures of HeLa cells, including a seπes of control solutions of unlabelled BSA, 5-ammophenyl-15- (hydroxyethylpyndiniumyl) poφhyrin (HEPP-NH2, amino precursor of HEPP-NCS), and PBS on its own Cells were seeded onto coverslips in 35 mm dishes.

Fluorescence images of cells were obtained with a Bio-Rad Radιance2000 confocal laser scanning microscope (Bio-Rad Microscience, Cambπdge, MA) on an inverted Olympus 1X70 microscope using a 60x (NA 1.4) oil immersion objective lens. The illumination source was the 514 nm line from a 25 mW argon ion laser. Poφhyπns were visualised with a 514 nm band-pass excitation filter, a 510 nm dichroic mirror, and a 570 nm long-pass emission filter.

Each field of cells was sectioned 3-dιmensιonally by recording images from a seπes of focal planes. Movement from one focal plane to another was achieved by a stepper motor attached to the fine focus control of the microscope, the step sizes (in the range 0.5 μm to 1 25 μm) being chosen with regard to the aperture size being used, so that there would be some overlap between adjacent sections. Enough vertical sections were taken so that the tops and bottoms of all the cells in each field would be recorded. Each image collected was the average of four scans at the confocal microscope's normal scan rate. During each imaging session calibration images were taken of: (l) a microscope slide containing medium, in order to measure background levels; (n) a slide containing ITC poφhyrin HEPP-NCS dissolved in DMSO; and (in) a slide beaπng only un-probed HeLa cells

Image data acquisition and remote microscope operation was earned out using the Bio-Rad Lasershaφ2000 software. All images were managed using Confocal Assistant version 4.02, (build 101) 1994-1996 Todd Clark Brelje. Artificial colour was applied using standard Bio-Rad look-up tables (LUT).

A preliminary evaluation of the fluorescence of HEPP-NCS at each of the excitation laser lines available on the CLSM set-up was earned out for a 0.01 mM solution of OH6 in DMSO. Figure 14 shows the UV-visible spectrum of HEPP-NCS identifying its pπncipal absoφtion bands. Unfortunately, no laser line was available in order to excite HEPP-NCS at its Soret band λ_max. Figure 15 demonstrates the relative intensities of fluorescence emission for HEPP-NCS when excited at 422 nm (optimal), and at the four wavelengths of the argon ion laser, 457, 476, 488, and 514 nm.

It was determined that the intensity of fluorescence emitted by a solution of HEPP-NCS when excited at 514 nm was roughly three times greater than fluorescence emission at excitation wavelengths of 457, 476, and 488 nm. The UV-visible absoφtion spectrum of HEPP-NCS showed that the 516 nm argon-ion laser line was the only excitation source compatible with HEPP-NCS. The three strongest laser lines, 457, 476, and 488 nm all excited in the region between the Soret and first Q band of HEPP-NCS, whereas the 514 line overlapped well with the Q band at 516 nm.

Cell cultures separately incubated with conjugate HEPP-NCS-BSA and each of the three controls, were subsequently washed and fixed. Coverslips containing the incubated cells were then cautiously mounted onto standard glass microscope slides ready to be imaged. All four argon-ion laser lines were tested, but, as expected satisfactory resolution of fluorescence could only be achieved using the 514 nm laser line.

A Z-series fluorescence image of HeLa cells incubated with HEPP-NCS-BSA is shown in Figure 16 (this Figure should be viewed from top left to bottom right). Consecutive sections were scanned with a 2μM step between each focal plane resolved by the microscope, thus enabling three dimensional visualisation of the localisation of the conjugate within the cell. Clearly the conjugate HEPP-NCS-BSA had entered the cell, no studies of the nature of cellular uptake were conducted, however it is most likely that uptake had taken place via endocytosis. It can be seen that the conjugate has not entered the nucleus and appears to be largely distributed throughout the cytoplasm.

When imaged, cells incubated with the BSA control or the PBS control, showed only very low, barely detectable levels of fluorescence, attributed to normal levels of cellular autofluorescence. No fluorescence was found to emanate from inside the cells, instead it appeared that the majority of HEPP-NH2 had become localised on the plasma membrane. Evidently the BSA component of the conjugate is required in order to facilitate the transport of poφhyrin to the interior of the cell. In summary, it has been shown that the cellular localisation of poφhyrin-BSA conjugates, constructed via the formation of covalent thiourea linkages, can be imaged using conventional CLSM techniques. Unconjugated poφhyrin HEPP-NH2 was not found to penetrate the cellular membrane, whereas a significant level of fluorescence was detected from inside cells incubated with HEPP-NCS-BSA, indicating good conjugate penetration.

Claims

1 A poφhyrin, chlorin or bacteriochlorin chromophore, which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein so as to enable delivery of the chromophore to a selected biological target in vitro or in vivo, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is such that on canying out conjugation of said chromophore to said protein by way of incubating said chromopiiore and said protein for one hour under standard protein conjugation conditions in J 0% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 5% or less.

2 A chromophore as claimed in claim 1, wherein said percentage of non-covalently bound chromophore out of total protein-bound chromophore is 4% or less; more preferably 3% or less, still more preferably 2% or less, even still more preferably 1% or less; most preferably 0-0.5%.

3 A poφhyrin, chlorin or bacteriochlorin chromophore, which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is sufficient to ensure that on canying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non- covalently bound chromophore out of total protein-bound chromophore is 20% or less, wherein said protein is bovine serum albumin.

4 A chromophore as claimed in claim 3, wherein said percentage of non-covalently bound chromophore out of total protein-bound chromophore is 18% or less; more preferably 15% or less; still more preferably 13% or less; yet more preferably 10% or less; even still more preferably 7% or less; even yet more preferably 5% or less; most preferably 3% or less.

5 A chromophore as claimed in any of claims 1-4, wherein said protein is a delivery protein with specific affinity for said biological target.

6 A chromophore as claimed in claim 5 wherein said biological target is a cell or a membrane, characterised in that said delivery protein possesses specific affinity for a receptor or channel exposed on the surface of said cell or membrane, which receptor or channel is adapted to cause or allow the passage of a molecule bound thereto across said membrane or into said cell.

7 A chromophore as claimed in any of claims 1-4, wherein said protein is a bridging polypeptide, which bridging polypeptide is adapted to be bound or linked to a complementary bridging polypeptide, which complementary bridging polypeptide can be bound or linked to said biological target or to a delivery protein with specific affinity for said biological target so as to enable delivery of said protein molecule to said biological target.

8 A chromophore as claimed in claim 7, wherein said bridging polypeptide and said complementary bridging polypeptide respectively comprise either one of avidin/streptavidin and biotin, or one of calmodulin and calmodulin binding peptide.

9 A chromophore as claimed in any preceding claim, wherein one or more of said hydrophilic meso substituents comprises a charged substituent, such as a zwitterionic substituent possessing both positively and negatively charged moieties, other than:

or

tionic substituent having a net positive charge, other than:

or an anionic substituent, having a net negative charge.

10 A chromophore as claimed in any preceding claim, wherein said hydrophilic substituent comprises a quartenised pyridyl (pyridiniumyl) ring.

11 A chromophore as claimed in claim 10, wherein said pyridiniumyl ring is linked by a carbon atom in said ring to said poφhyrin, chlorin or bacteriochlorin chromophore core and comprises one quartenising ring substituent Q which is N-linked to said pyridiniumyl ring for quartenising said nitrogen atom, which quartenising ring substituent Q comprises ethyl, or branched or linear propyl, butyl, pentyl, hexyl, heptyl or octyl, or aryl such as phenyl, or heteroaryl such as pyridyl, or a hydrophilic group W; which hydrophilic group W comprises a glycosyl group, or an amino acid, or said group W is a group selected from R₃L, or YιR₃, or (R₄YιR₅)_x, or

or Y₁R₄Y₂R₅, or -COO^", or -S0₃\ or hydroxy, or oxo; wherein R₃ is methyl or ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, or phenyl, or pyridyl, or pyridiniumyl, or a group R₆R₇, where R₆ is methyl or ethyl and R₇ is phenyl or pyridyl or pyridiniumyl; where R₃ does not comprise pyridiniumyl, L represents one or more groups selected from -OH, -COO^" (or -COOH) or -S0₃ ^"(or S0₃H); where R₃ comprises pyridiniumyl, L represents one or more groups selected from -OH, -COO^" (or -COOH), - S0₃ ^" (or S0₃H), or hydrogen; Y_\ and Y₂ are independently selected from O and S; R is selected from a single bond, methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, phenyl, pyridyl, and pyridiniumyl; R₅ is selected from hydrogen, methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, phenyl, pyridyl, and pyridiniumyl; and x is an integer from 1 to 5.

12 A chromophore as claimed in claim 10, wherein said pyridiniumyl ring is substituted one or more times by one or more hydrophilic groups W as defined in claim 1 1.

13 A chromophore as claimed in claim 11 or claim 12, wherein said R₃ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, and said L includes a terminating hydroxy, carboxy or sulfonate group.

14 A chromophore as claimed in claim 11 or claim 12, wherein said hydrophilic group W is a group -(CH₂)_aOH, or -(CH₂)_aCOO^" (or -(CH₂)_aCOOH), or -(CH₂)_aS0₃ ^" (or -(CH₂)_aS0₃H) where a is 1, 2, 3, 4, 5, 6, 7 or 8; most preferably 1, 2 or 3.

15 A chromophore as claimed in claim 11 or claim 12, wherein said Yi is O, and said x is greater than 1.

16 A chromophore as claimed in claim 11 or claim 12, wherein said hydrophilic group W comprises a glycosyl group which is a sugar such as glucose, mannose, maltose, or a thiosugar such as thiogalactopyranose, thioglucopyranose or thiomannopyranose.

17 A chromophore as claimed in claim 11 or claim 12, wherein said hydrophilic group W comprises an amino acid selected from lysine, cysteine, tyrosine, aspartate, glutamate, serine and threonine.

18 A chromophore as claimed in any of claims 11-17, wherein said hydrophilic group W further comprises one or more hydroxy or oxo substituents.

19 A chromophore as claimed in any of claims 1-9, wherein said hydrophilic substituent comprises a quartenised amine group -F^QiQ^Qs or a quartenised phosphonium group -P⁺QιQ₂Q_3. each of which Qi, Q2 and Q₃ is selected from methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, aryl such as phenyl, heteroaryl such as pyridyl, and a hydrophilic group W as defined in any of claims 11-18.

20 A chromophore as claimed in claim 19, wherein said Qi, Q and/or Q₃ is substituted by a hydrophilic group W as defined in any of claims 11-18.

21 A chromophore as claimed in claim 19 or claim 20, wherein at least one, more preferably two, most preferably each of said Qi, Q2 and Q₃ comprises an uncharged aryl or heteroaryl moiety, such as a phenyl ring, naphthyl ring, anthracene ring or a pyridyl ring.

22 A chromophore as claimed in any of claims 1-9, wherein said hydrophilic substituent comprises a phosphate group -P(0)(OR )(O^") or a phosphonate group - OP(0)(OR₇)(O^"), wherein said R₇ is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl.

23 A chromophore as claimed in any preceding claim, wherein said hydrophilic group is linked to the core of said poφhyrin, chlorin or bacteriochlorin chromophore by way of a linking group L₂, which linking group L₂ either comprises a group -Rp or - R₁R₂-, where each of Ri and R₂ is independently selected from a single bond, or methyl, or phenyl, or branched or linear ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl; or said linking group L₂ comprises an ether or thioether chain such as a chain -A1R1 A2R₂-, where each of A] and A₂ is independently selected from a single bond or S or O, and Ri and R₂ are as hereinabove defined, or a polyether or polythioether chain based on repeating units of said -A1R1 A2R2-. such as a C₂-₃o polyethylene glycol chain; or said linking group L₂ is a group RιR₂ wherein said Ri is phenyl and said R₂ is methyl or ethyl or propyl; or said linking group L₂ is a group Ri wherein Ri is a single bond. 24 A chromophore as claimed in any of claims 1-9, wherein said hydrophilic substituent comprises or consists of a group R11R12R-3, where Rn is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, R₎₂ is NH, O, S or CH₂, and R₎₃ is hydrogen or a hydrophilic group W as defined in any of claims 11-17.

25 A chromophore as claimed in claim 24, wherein said R11R₁₂R13 constitutes a polyhydroxyalkyl, preferably a dihydroxyalkyl.

26 A chromophore as claimed in any preceding claim, wherein said conjugating group Z comprises a group which is capable of bonding covalently to an amine group on a polypeptide molecule; such as an isocyanate, isothiocyanate, or NHS ester group.

27 A chromophore as claimed in any preceding claim, which is a poφhyrin chromophore of formula (I) below:

or a chlorin chromophore of any of formulas (II), (III), (IV), or (V) below:

(ID (III)

or a bacteriochlorin chromophore of any of formulas (VI) and (VII) below:

(VI) (VII)

wherein Rio is or comprises said conjugating meso substituent; at least one of R20, R₃0 and R₄o is or comprises said hydrophilic meso substituent; and each of Xi, X2, X3 and X is independently selected from H, OH, halogen, Cι-₃ alkyl and OCι-₃ alkyl, or X] and X and/or X₃ and X together form a bridging moiety selected from O, CH₂, CH C₁.₃ alkyl, or C(Cι-₃ alkyl) , such that X* and X₂ and or X₃ and X₄ with the adjacent C-C bond form an epoxide or cyclopropanyl structure.

28 A set of fluorochromic markers for multicolour fluorochromic analysis, comprising at least two chromophores selected from the group consisting of a poφhyrin chromophore, a chlorin chromophore and a bacteriochlorin chromophore, each of which chromophores is a chromophore in accordance with any preceding claim and each of which chromophores comprises the same 5, 10, 15 and 20 meso substituents.

29 A kit comprising a chromophore in accordance with any of claims 1-27 or a set of chromophores in accordance with claim 28, wherein said chromophore or each chromophore is conjugated to a bridging polypeptide that is adapted to bind to a complementary bridging polypeptide so as to couple the chromophore to said complementary bridging polypeptide; and a construct or plurality of constructs each of which comprises said complementary bridging polypeptide fused or coupled to a delivery protein which is adapted to bind specifically to said biological target; the anangement being such that said chromophore or each chromophore in the kit is adapted to bind to a different construct in the kit with specificity for said specific biological target, so as to link said or each chromophore to a delivery protein with specificity for said specific biological target.

30 A method for attaching a chromophore in accordance with any of claims 1-27 or a set of chromophores in accordance with claim 28 to said specific biological target or targets; comprising the steps of providing a kit in accordance with claim 29 and introducing the components of said kit into the vicinity of said specific biological target or targets, under conditions suitable for enabling the binding of said or each delivery protein to said specific biological target or targets.

31 A method for fluorescence-activated sorting of target cells from a mixture of cells, comprising the step of attaching to said target cells a chromophore in accordance with any of claims 1-27 or a set of chromophores in accordance with claim 28, illuminating said mixture of cells so as to cause fluorescence of one or more of said chromophores attached to said target cells, imparting a charge to the fluorescing cells, and passing said mixture of cells through a polarised environment so as to cause or allow said charged cells to be separated from said mixture.

32 A method for the visualisation and/or counting of a plurality of target cells, said target cells including cells of two or three different cell types, comprising the steps of providing a chromophore set in accordance with claim 28, which chromophore set comprises two or three chromophores each of which is adapted to be delivered to a different one of said cell types; attaching said chromophores in the set to said target cells in accordance with the method of the present invention; illuminating said target cells so as to cause the emission of fluorescence by said chromophores; detecting the fluorescent emission bands produced by each of said chromophores; and optionally measuring for each of said bands the area under an emission/wavelength curve, so as to obtain a measure of the number of fluorescent cells of each respective cell type.

33 A method for causing the death of a target cell, comprising the step of attaching a chromophore in accordance with any of claims 1-27 to said cell and illuminating said cell so as to cause the production of singlet oxygen in the vicinity of said cell, thereby causing the death of the cell.

34 A method for treating a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), comprising the step of administering to a patient in need thereof an effective amount of a chromophore in accordance with any of claims 1-27, which chromophore is adapted to be targeted to a target cell specific molecule on the surface of said diseased or undesired cells for attachment thereto, such that the chromophore is caused to be attached to said cells, and illuminating said cells with light so as to cause the production of singlet oxygen in the vicinity of said cells, thereby killing said cells.

35 A pharmaceutical composition for administration to a patient for the treatment of a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), which composition comprises a chromophore in accordance with any of claims 1-27 that is adapted to be delivered to said diseased or undesired cells, and a suitable carrier.

36 A chromophore in accordance with any of claims 1-27 for use in the treatment of patients suffering from a disease or disorder which is characterised by the presence in the body of diseased or undesirable cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD); said chromophore being adapted for delivery to said diseased or undesired cells.

37 A method for synthesising a sugar-substituted meso-aryl poφhyrin/chlorin/bacteriochlorin, comprising the steps of providing a meso-aryl poφhyrin/chlorin/bacteriochlorin in which the meso-aryl substituent is substituted with a leaving group and at least one electron withdrawing group, and reacting this poφhyrin/chlorin/bacteriochlorin with a nucleophilic sugar such as to displace the leaving group on the meso-aryl substituent by way of a nucleophilic substitution reaction.

38 A method as claimed in claim 37, wherein said nucleophilic sugar is a deprotonated sugar such as deprotonated glucose, mannose, or maltose, having an anomeric hydroxyl group, which anomeric hydroxyl group is deprotonated. 39 A method as claimed in claim 37, wherein said nucleophilic sugar is a thiosugar, such as thiogalactopyranose, thioglucopyranose or thiomannopyranose.

40 A method as claimed in any of claims 37-39, wherein said meso-aryl substituent is a phenyl substituent which is tri- or penta-substituted by fluoro.

41 A method as claimed in any of claims 37-40, wherein said sugar comprises one or more protecting groups, further comprising the step of removing protecting groups from said sugar.

42 A method as claimed in any of claims 37-41, further comprising the step of dissolving said sugar-substituted poφhyrin, chlorin or bacteriochlorin in tetrahydrofuran or dichloromethane, and forming an NCS group on a meso-aryl substituent of said sugar- substituted poφhyrin, chlorin or bacteriochlorin, in order to obtain a sugar-substituted NCS-linked meso-aryl poφhryin, chlorin or bacteriochlorin.