NZ523011A

NZ523011A - Improvements in and relating to chromophores such as porphyrin, chlorin and bacteriochlorin chromophores

Info

Publication number: NZ523011A
Application number: NZ523011A
Authority: NZ
Inventors: Ross William Boyle; Oliver James Clarke; Jonathan Mark Sutton; John Greenman
Original assignee: Wellcome Trust Ltd
Priority date: 2000-06-26
Filing date: 2001-06-26
Publication date: 2004-07-30
Also published as: AU783640B2; CA2414089A1; WO2002000662B1; WO2002000662A1; JP2004501923A; EP1294726A2; AU7074801A

Abstract

Porphyrin and porphyrin-based chromophores and sets of porphyrin and porphyrin-based chromophores such as porphyrin, chlorin and bacteriochlorin chromophores, which may be particularly useful in a range of photodynamic applications, including photochemotherapy and fluorescence analysis and imaging are disclosed. Also disclosed are methods for the production of such chromophores and methods for the use of such chromophores in analysis and in medicine.

Description

523011 WO 02/00662 PCT/GB01/02846 IMPROVEMENTS IN AND RELATING TO CHROMOPHORES The present invention relates to novel porphyrin and porphyrin-based chromophores and sets of porphyrin 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 porphyrin and porphyrin-based chromophores both as research tools, for example in fluorescence-activated cell sorting (FACS), and as therapeutic agents in photodynamic therapy (PDT) f6r bringing about the death of targeted cells in vivo, is widely 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.

Evidently, for the purposes of fluorescence imaging or analysis, or 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 0. lum 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.

Hitherto, however, few attempts have been made to control the targeting of porphyrin photosensitisers to particular target cells in vivo for the purposes of PDT. Instead, reliance has typically been placed on the inherent tendency of porphyrins to 1 accumulate in tumours in the absence of lymphatic drainage from tumour structures. Photofrin®, a photosensitising agent comprising a mixture of porphyrin structures derived from hematoporphyrin-IX by treatment with acids which is commercially used in the treatment of carcinomas and sarcomas, is, for example, conventionally administered systemically to patients without any targeting vehicle or means. This is evidently undesirable, as incorrect localisation of the photosensitiser will not only decrease the efficiency of the photochemotherapy, but may also result in the death of healthy cells.

Efforts have been made to achieve the specific attachment of chromophores to biological targets in vitro, in particular for the purposes of FACS and fluorescence imaging, by covalently conjugating the chromophores to suitable protein delivery molecules. This approach has however been subject to various problems. Firstly, the degree of background fluorescence caused by non-specific binding of porphyrin chromophores to cell surfaces has proved difficult to reduce. Secondly, it has been found that the attachment of a chromophore to a protein molecule can result in a significant degree of excited state quenching by the proximate protein, which will clearly reduce the efficacy of the chromophore as a marker or in targeted photodynamic applications.

A reduction in these effects remains a desirable objective.

According to one aspect of the present invention therefore, there is provided a porphyrin chromophore of formula (I) below: R.

(I) or a chlorin chromophore of any of formulas (II), (III), (IV), or (V) below: 2 (II) (HI) (IV) or a ^o«nctaophoreofMsoffmiiii (VI) and (VH) below: 7 (VI) =N21 2, N-22 H /8 V ~~~N24 23 N- \1 ) R2 13 /12~ X4 wherein Ri is a phenyl group linked R-2 is py] R4P+(R5)(R6)(R7) _ „ -id to a conjugating group Z, Z is -NCS; R2 is pyridiniumyl or phenyl substituted by -0(Ci-C6 alkyl), pyridiniumyl n> ACT) S- or I INTELLECTUAL PkO? £RTV OFFICE OF N.Z. 27 MAY 203*1 WO 02/00662 PCT/GB01/02846 R4 is a single bond or C1-C6 alkyl; each of R5, and R7 is independently hydrogen, C1-C6 alkyl or aryl optionally substituted by OH, C1-C6 alkyl, C1-C6 alkoxy, aryl, oxo, nitro, amino or cyano; R3 is H, C1-C6 alkyl (which may optionally be substituted by one or more halogen or OH groups) or an R2 group as defined above; each of Xi, X2, X3 and Xjis independently selected from H, OH, halogen, C1.3 alkyl and OC1.3 alkyl, wherein each of Ri, R2 and R3 (when an R2 group) is optionally further substituted one or more hydrophilic substituents selected from -OH, -CN, -NO2, halogen.

It has been found that the inclusion of one or more hydrophilic substituents around the core of a 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 ceils or tissues, for example for the purposes of PDT or FACS, a decrease in non-covalent binding between the chromophore and the protein will reduce the degree of nonspecific transfer of chromophore to cell surfaces, which will substantially increase the accuracy of targeting the chromophore to the cells or tissue of interest.

Accordingly, according to yet another aspect of the present invention 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 as described above, illuminating said mixture 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 the mixture of cells through a polarised environment so as to cause or allow said charged cells to be separated from said mixture.

In some embodiments, each or some of Xi- X4 is H. In particularly preferred embodiments, however, each of Xi - X4is 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 porphyrins, owing to the presence of extra hydrophilic hydroxy groups around the core of the chromophore.

Preferably, said aryl moiety Ri may 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 C 1.6 branched or linear alkyl chain. 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 porphyrin, 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(Ci-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 Ri by a single bond. Alternatively, said conjugating group Z may be linked to said aryl moiety Ri 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 WO 02/00662 PCT/GB01/02846 lOOOgmol"1. In particular, said linking moiety may comprise an anthracene, acridine, anthranil, naphthyl 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 hydrophilic or amphiphilic moiety of the kind described above, such as a C2-C30 polyethylene glycol moiety. 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 Ri may be further substituted by one or more hydrophilic substituents, such as hydroxy, which will serve to improve the hydrophilicity of said chromophore.

Said hydrophilic aryl moiety R2 may comprise a phenyl ring, which phenyl ring may be substituted one or more times, preferably at least two times, by one or more hydrophilic substituents which serve to increase the hydrophilicity of said aryl moiety R2. Said 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 C1.6 branched or linear alkyl chain. Alternatively, said hydrophilic aryl moiety R2 may comprise a heteroaryl ring, such as a pyridyl or quaternised pyridyl (pyridiniumyl) ring, which heteroaryl ring may be substituted one or more times, preferably at least two times, by one or more hydrophilic substituents which serve to increase the hydrophilicity of said aryl moiety R2. Said heteroaryl ring may preferably be linked by a single bond to the macrocyclic core of said chromophore or may alternatively be linked thereto by a Q.6 branched or linear alkyl chain. Said one or more hydrophilic substituents may advantageously be selected from hydroxy; alkoxy such as methoxy or ethoxy; C2-C15 polyethylene glycol; quatenised pyridyl (pyridiniumyl) such as N-methylpyridiniumyl; mono-, di- or poly-saccharide; Ci. galkylsulfonate; a phosphonium group R4P(R5)(R<;)(R7), wherein R4 is a single bond or Ci-g alkyl, and each of R5, Re and R7 is independently selected from hydrogen, an aryl ring such as a phenyl ring, a heteroaryl ring such as a pyridyl ring, and a C 1.6 alkyl chain, which aryl ring, heteroaryl ring or Ci.6 alkyl chain is unsubstituted or is substituted one or more times by hydroxy, Ci.g alkyl or alkoxy, aryl, oxo, halogen, nitro, amino or cyano; or a phosphate or phosphonate group Rg0P(0)(0R9)(0Rio) or RgP(O)(OR9)(ORi0) respectively, wherein Rg is a single bond or alkyl, and each of R9 and Rio is independently selected from hydrogen and Ci-6 alkyl. Preferably, each of said R5, Rg and R7 may be the same, and may advantageously be unsubstituted phenyl. Suitably, said Rs may be methyl. Advantageously, said R9 and said Rio may be the same, and/or may be methyl or ethyl.

In especially preferred embodiments, said hydrophilic aryl moiety R2 is selected from m,m-(dihydroxy)phenyl or a PEGylated derivative thereof; m,m,p-(trihydroxy)phenyl OH or a PEGylated derivative thereof; o,p,o-(trihydroxy)phenyl OH or a PEGylated derivative thereof; m- or p-((Ci_ 6)alkyltriphenylphosphonium)phenyl such as p-(methyltriphenylphosphonium)phenyl PPh. + m- or p-(Ci.6alkylphosphono-di-alkoxy)phenyl such as p-methylphosphono-di-ethoxy)phenyl PCT/GBO1/02846 BO OB m- or p-(Ci-6alkylphosphonato-di-alkoxy)phenyl such as p-methylphosphonato-di-ethoxy)phenyl Eto' "oEt m- or p-(N-methyl-pyridiniumyl)phenyl and meta- or para- sugar-substituted phenyl such as pentose-, hexose- or disaccharide-substituted phenyl In other preferred embodiments, said hydrophilic aryl moiety R2 comprises a quaternised pyridyl (pyridiniumyl) group such as a p-N-(Ci.6alkyl)pyridiniumyl group or m-N-(Ci-6alkyl)pyridiniumyl group. Quaternised pyridyl (pyridiniumyl) groups are highly hydrophilic and display advantageous properties when incorporated into chromophores in accordance with the invention. Particularly preferred groups in this regard are m- or p-N-((Ci^)alkyl)pyridiniumyl, such as m-N-methylpyridiniumyl 8 PCT/GBO1/02846 CH3 or p-N-methylpyridiniumyl In other especially preferred embodiments, said quaternised pyridiniumyl group may comprise a zwitterionic group, such as p-N-(C].6alkylsulfonate)pyridiniumyI or m-N-(C\.6alkylsulfonate)pyridiniumyL; in particular, p-N-(propylsulfonate)pyridiruumyl Preferably, the or each quaternised pyridiniumyl group Ra may be associated with a halide counterion, such as an iodide counterion .or, in most preferred embodiments, a chloride counterion.

In some advantageous embodiments, R3 is H, such that said chromophore constitutes a 5,15-diaryl-porphyrin, -chlorin or -bacteriochlorin. In other advantageous embodiments, said R3 is a hydrophilic aryl or non-aromatic moiety. For example, said R3 may comprise a hydrophilic aryl moiety as defined above in relation to R2. Said hydrophilic aryl moiety R3 may be the same as said hydrophilic aryl moiety R2, such that the chromophore possesses the same substituents at the 10, 15 and 20 positions thereof; or may be different from said hydrophilic aryl moiety R2. Alternatively, said R3 may comprise a hydrophilic alkyl moiety, such as a Cj-g alkyl chain which is substituted one or more times by one or more hydrophilic substituents such as hydroxy or C2-15 polyethylene or m-N-(propylsulfonate)pyridiniumyl 9 glycol. In particularly preferred embodiments, said R3 comprises polyhydroxy(Ci-6 alkyl), such as 1,2-dihydroxyethyl.

Chromophores in accordance with the invention wherein R2 is the same as R3 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 porphyrins from dipyrromethanes (Arsenault et al, J.

Chew. Soc. I960, 82:4384-4389 - incorporated herein by reference) .

A generalised scheme for the synthesis of 5-isothiocyanatophenyl-15-pyridinium porphyrins, chlorins and bacteriochlorins in accordance with the present invention is set out as Scheme 1 below, in which "RX" represents a quatemising group such as Ci-6 alkyl or a hydrophilic substituent as defined above in relation to formulas (I) to (VII): WHFmoc NHFmoc (i) Piperidine (ii) TOP (iii) RX (i) OsCtypyridine WH2S iHFmoc (i) Piperidine (li> TDP (iii) RX (i) Piperidine (ii) TDP (iii) RX Scheme 1 A generalised scheme for the synthesis of 5-isothiocyanatophenyl-15-methylphosphoniumphenyl porphyrins, chlorins and bacteriochlorins in accordance with the present invention is set out as Scheme 2 below, wherein R represents hydrogen, Ci^ alkyl, a heterocyclic group or an aromatic group : NHBoc NHBoc NHBoc (i) CBr^PPhj (ii) PR 3 T (iii) TMSI NCS (i) 0s04/pyridine (ii) H,S (i) 0s04/pyridine OD HjS (i) CBrJPPh, (ii) PR3 (iii) TMSI (iv) TDP (0 CBr^PPh (ii) PR3 (in) TMSI (iv) TDP NCS Scheme 2 Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein said R2 and optionally said R3 comprises pyridiniumylphenyl may be synthesised in accordance with the generalised reaction scheme set out below as 11 WO 02/00662 PCT/GB01/02846 Scheme 3, wherein "R" represents hydrogen or one or more hydrophilic substituents as defined above in relation to formulas (I) to (VII); Scheme 3 Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein said R2 and optionally said R3 comprise alkylphosphonatophenyl or alkylphosphonophenyl may be synthesised in accordance 12 PCT/GBO1/02846 with the generalised reaction scheme set out below as Scheme 4, wherein "R" represents OH, ONa, or 0(Ci-6 alkyl): Scheme 4 In a further aspect of the invention, there is provided a novel method for the synthesis of a 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore having selected substituents at the 5, 10, 15 and 20 meso-positions thereof; comprising the steps of providing a 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore; attaching a leaving group Q to the 10 and 20 meso-positions of said chromophore, which leaving group Q is selected from halide and triflate; providing a coupling reagent (RnO)(RnO)BRi3, wherein Rn and Ru are independently selected from H or Ci-6 alkyl, or Ri i and R12 together constitute a C1.6 alkyl chain bridging said two O atoms, and R13 is vinyl or aryl, such as a hydrophilic aryl moiety as 13 hereinbefore defined in relation to R3; and reacting said chromophore with said coupling reagent in the presence of a base selected from potassium phosphate, sodium phosphate, caesium carbonate and barium hydroxide, and a Pdo catalyst; such that said R13 replaces said leaving group Q at the 10- and 20- meso positions of said chromophore.

Pdn-catalysed Suzuki coupling reactions using boronic acid or boronic ester reagents are known in the art, and are described for example in Miyaura & Suzuki, Palladium-catalyzed cross-coupling reactions of organoboron compounds, Chem. Rev. (1995) 95:2457-2483; the disclosure of which is incorporated herein by reference.

Hitherto, however, attempts to carry out Suzuki-coupling at the meso-positions of porphyrins, chlorins or bacteriochlorins, as a means of importing selected substituents onto said meso-positions, have failed. The present inventors have found however that under the reaction conditions of the invention, Suzuki-coupling proceeds rapidly and successfully at the 10- and 20- meso-positions of the starting porphyrin, chlorin or bacteriochlorin chromophore. This method thereby enables convenient synthesis oftetra-meso-substituted porphyrins, chlorins or bacteriochlorins by Suzuki-coupling.

Said leaving group Q may be chloride, bromide, iodide or triflate (trifluoromethanesulfonate). Suitably, said leaving group Q may be bromide. Methods for the meso-bromination of di-meso-substituted porphrins, chlorins or bacteriochlorins are known in the art. For example, said 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be halogenated at the 10- and 20- meso-positions thereof by way of reaction with halosuccinimide, such as bromosuccinimide.

Said coupling reagent may comprise a boronic ester or a boronic acid. In preferred embodiments, each of said R11 and R12 is H, such that said coupling reagent is a boronic acid.

Advantageously, said 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore is a chromophore in accordance with the invention, or a protected form thereof. Thus, said 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be selected from a porphyrin chromophore of formula (VIII) below: 14 (VIE) or a chlorin chromophore of any of formulas (IX), (X), (XI), and (XII) below: X2\ f >-nh // 22H 2t/ 21 19/T-N24 2N3r^1° 1V~N (IX) (X) (XH) or a bacteriochlorin chromophore of any of formulas (XDT) and (XIV) below: PCT/GBO1/02846 X k3 (XIII) (XIV) wherein R4 is a group Ri as defined above in relation to formulas (I) to (VII) or a protected form thereof or a group convertible thereto; R5 is a group R2 as defined above in relation to formulas (I) to (VII) or a protected form thereof or a group convertible thereto; and each of Xi, X2, X3 and X4 is independently selected from H, OH, halogen, C1-3 alkyl and OC1.3 alkyl, or Xi and X2 and/or X3 and X4 together form a bridging moiety selected from O, CH2, CH C1.3 alkyl, or C(Ci.3 alkyl)2, such that Xi and X2 and/or X3 and X4 with the adjacent C-C bond form an epoxide or cyclopropanyl structure.

Accordingly, where R13 is a hydrophilic aryl substituent as defined above in relation to R3, said 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may also constitute a chromophore in accordance with the present invention.

Said Pd0 catalyst may, for example, comprise Pd(PPh3)4, PdCl2(PPh3)2, or Pd(OAc)2. Advantageously, said Pd0 catalyst may comprise Pd(PPh3)4.

Said coupling reaction is performed in a solvent, which may be selected from toluene or dry THF. It is found that the coupling reaction proceeds swiftly in dry THF, and so dry THF is preferred as solvent.

Optionally, where said Rl3 is vinyl, said 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be subjected following said coupling reaction to an osmylation reaction utilising OSO4, such as to convert said 10-and 20- vinyl substituents to hydroxyalkyl. Said osmylation reaction may be carried out under conditions identical to those suitable for converting a porphyrin to a di-beta-hydroxy-chlorin and then to a tetra-beta-hydroxy-bacteriochlorin. It is noted that this step 16 may be performed in accordance with the invention on 5,10(vinyl),15,20(vinyl)-meso-substituted porphyrin, chlorin or bacteriochlorin chromophores which are obtained otherwise than in accordance with the method of the invention, such as by way of Pd-catalysed Stille coupling performed on said 5,15-di-meso-substituted chromophore in accordance with the method described in DiMagno et al, J. Org. Cham. 1993:58, 5983-5993, (incorporated herein by reference) wherein vinyl tributyl tin is used as a coupling reagent.

Where said tetra-meso-substituted chromophore is a porphyrin or a chlorin' chromophore, said chromophore may be respectively converted to a chlorin or bacteriochlorin chromophore or to a bacteriochlorin chromophore in accordance with methods known to the man skilled in the art. For example, said porphyrin or chlorin chromophore may be osmylated by way of reaction with OsC>4, such as to produce a di-beta-hydroxy-chlorin or a tetra-beta-hydroxy-bacteriochlorin.

Generalised schemes for reactions in accordance with the present invention are set out in Schemes 5 and 6 below. In Scheme 5, "R" and "Rj" each represents one or more hydrophilic substituents as defined above in relation to R2 and R3 respectively. In Scheme 6, "R" represents one or more hydrophilic substituents as defined above in relation to R2, and "X" represents a carbon or nitrogen atom.

Br Br (i) Pd(PP^)4/K3P04 NHBoc R R Scheme 5 17 HFmoc HO- HO PHN (|) OsCVpyridine (3 eq) \\ (ii) H2S -OH (i) piperidine (ii)TDP (i) OsCVpyridine (2eq) WHjS (iii) piperidine t (iv)TDP IslCS (i) piperidine (ii)TDP Scheme 6 According to another aspect of the present invention, there is provided a 5,15-diphenylporphyrin, 5,15-diphenylchlorin or 5,15-diphenyibacteriochlorin chromophore, wherein each of the ortho-, meta-, and/or para- positions of each of the 5- and 15- phenyl groups is substituted by a substituent P1-P5 and Q1-Q5 respectively which is independently H or an inert substituent which in combination with the other substituents P1-P5 and Q1-Q5 does not substantially impair the fluorescent properties of the chromophore; and the chromophore further comprises a. conjugating group Z which is capable of conjugating the chromophore to a polypeptide molecule for delivering said chromophore to a specific biological target in vitro or in vivo.

Such chromophores 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 targetted to a specific biological target, thus facilitating 18 PCT/GBO1/02846 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.

Advantageously, said fluorochrome is selected from the following compounds: ^3 ^3 .^3 wherein each of Xi, X2, X3 and X4 are as defined above in relation to the first aspect of the invention. Optionally, said chromophore may be further substituted at one or more of the 2, 3, 7, 8, 12, 13, 17 or 18 positions thereof by a C1.3 alkyl substituent.

In the foregoing chemical structures, Z has been omitted for clarity. However, said Z substituent may be attached to any of the 1-4, 6-14, or 16-20 positions of each chromophore, or may be one of the substituents Pi-Ps or Q1-Q5, or may be attached to one of the 5- or 15- 'phenyl groups through one of said substituents P1-P5 or Q1-Q5. 19 In some embodiments, each of P1-P5 is the same or substantially the same as the corresponding one of Q1-Q5, such that said two primary phenyl rings are symmetrically substituted. In other embodiments, one or more of P1-P5 is not the same as the corresponding one of Q1-Q5, such that said two primary phenyl rings are not symmetrically substituted. In particular, all of P1-P5 and/or all of Q1-Q5 may comprise H, such that one or both of said two primary phenyl rings is or are unsubstituted.

Advantageously, said substituents P1-P5 and Q1-Q5 collectively provide a degree of steric hindrance around the core of said chromophore which is sufficient to reduce the rate of spontaneous oxidation of said chromophore, such that said chromophore is substantially inert in air, but which does not to a substantial extent inhibit selective addition or substitution at the 2, 3, 7, 8, 12, 13, 17 or 18 positions around the core of said chromophore. Thus, each ofPi, P5, Qi and Qs may be H. Typically, the total cumulative molecular weight of said substituents P1-P5 does not exceed lOOOgmol"1, and the total cumulative molecular weight of said substituents Q1-Q5 does not exceed lOOOgmol"1.

One or more of said substituents P1-P5 and Q1-Q5 may comprise -OH, -CN, -NO2, halogen, -T or -OT, where T is a C1-C15 alkyl, cycloalkyl or aryl group or a hydroxylated, halogenated, sulphated or aminated derivative thereof or a carboxylic acid, ester, ether, polyether, amide, aldehyde or ketone derivative thereof One or more of said substituents P1-P5 arid Q1-Q5 may additionally or alternatively comprise a C3-C12 cycloalkyl and/or aryl ring structures, or between two and six, preferably two - three, fused or linked C3-C12 cycloalkyl and/or aryl ring structures, each of which ring structures may optionally comprise one or more N, O or S atoms. In particular, one or more of said substituents P1-P5 and Q1-Q5 may comprise a quatenised amine or pyridyl group, such as an N-methyl pyridyl (pyridiniumyl) group.

Preferably, one of P1-P5 and Q1-Q5 is a conjugating substituent which comprises said conjugating group Z. In particularly preferred embodiments, said conjugating substituent is P3 or Q3, such that said conjugating group Z is provided on the para-position of one of the two primary phenyl rings.

Suitably, said conjugating group is as defined above in relation to the first aspect of the invention.

PCT/GBO1/02846 In particular, one or more of said substituents P1-P5 and Q1-Q5, not being said conjugating substituent, may consist of a member independently selected from the group consisting of AjZjAm; wherein Z\ is Z2, Z2A5 or Z2A5A5; Aj and A5 are independently selected from -(CA2A3)n- , -C(Y)(CA2A3)n-, -C(Y)Y'(CA2A3)n-, -C(Y)NA4(CA2A3)n-, -NA4C(Y)(CA2A3)n~, -NA4(CA2A3)n, -YC(Y')(CA2A3)n-and -Y(CA2A3)n-; n = 0 - 6; Y and Y' are independently 0 or S; A2, A3 and A4 are independently H or Cj.2 alkyl which is unsubstituted or substituted by one or more fluorines; Ag = - (C2H40)m- or -S(0)p; m = 1 - 12; p = 0 - 2; Z2 is a single bond or Z3- Z3 is selected from Z4, Z5 and 25, wherein Z3 is unsubstituted or substituted one or more times by OH, halo, CN, N02, AjAjo, AgAg, NA^A^i, C(Y)Aj, C(Y)Y'A% Y(CH2)qY'A7, Y(CH2)qA7, C(Y)NA10An> Y(CH2)qC(Y')NAioAn, Y(CH2)qC(Y')A9, NAi0C(Y)NA10An, NA10CtY)An, NAi0C(Y)Y'A9, NA10C(Y)Z6, C(NA10)NAl0Ai b CfNCNJNAiQA] h C(NCN)SA9, NA10C(NCN)SA9, NAjoCCNC^NAjoA! J, NA10S(O)2A9, S(0)rA9, NA, 0C(Y)C(Y')NA10A11, NA10C(Y)C(Y')A10 or Z6; q = 0, 1 or 2; r - 0 - 2; A7 is independently selected from H and A9; Ag is O or A9; A9 is C 1.4 alkyl which is unsubstituted or substituted by one or more fluorines; A\q is OA9 or A^; Aj 1 is A7 or when A10 and A^ 1 are as NAjqAh they may together with the nitrogen form a 5 to 7 membered ring comprising only carbon atoms or carbon atoms and at least one heteroatom selected from O, N and S; Z4 is Cg_i2 aryl or aryloxyC 1.3alkyl; Z5 is selected from furanyl, tetrahydrofuranyl, indanyl, indenyl, tetrahydropyranyl, pyranyl, thiopyranyl, tetrahydrothiopyranyl, tetrahydrothienyl, thienyl, C3_g cycloalkyl or C4.8 cycloalkyl containing one or two unsaturated bonds, and Cj.\ 1 polycycloalkyl; Z5 is selected from N-azolyl, dioxadiazinyl, dioxadiazolyl, dioxanyl, 2-N-dioxatriazinyl, dioxazinyl, N-dioxazolyl, dioxolyl, dithiadiazinyl, dithiadiazolyl, N-dithiatriazinyl, dithiazinyl, N-dithiazolyl, 1-N-imidazolyl, N-morpholinyl, pyrollyl, tetrazolyl, thiazolyl, triazolyl, oxazinyl, oxazolyl, naphthydrinyl, oxadiazinyl, oxadiazolyl, oxatetrazinyl, oxatriazinyl, oxatriazolyl, oxazinyl, oxazolyl, pentazinyl, phthalazinyl, N-piperidinyl, 21 N,N-piperazinyl, 1-N-pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, tetrathiazinyl, tetrazinyl, 1-N-tetrazolyl, tetroxazinyl, thiadiazinyl, thiadiazoyl, thiatetrazinyl, thiatriazinyi, thiatriazolyl, thiazolyl, triazinyl, 1-N-triazolyl, trioxadazinyl, trioxanyl, trioxazinyl, trioxazolyl, trithiadiazinyl, trithiazinyl, trithiadiazolyl; wherein Z4, Z5 or Zg may be fused to one or more other members selected independently from Z4, Z5 and Zg; Ah is hydrogen, methyl, hydroxyl, aryl, halo substituted aryl, aryioxyCi-3 alkyl, halo substituted aryloxyCi-3 alkyl, indanyl, indenyl, C7.11 polycycloalkyl, tetrahydrofuranyl, furanyl, tetrahydropyranyl, pyranyl, tetrahydrothienyl, thienyl, tetrahydrothiopyranyl, thiopyranyl, C3-6 cycloalkyl, or a C4-6 cycloalkyl containing one or two unsaturated bonds, wherein the cycloalkyl or heterocyclic moiety is unsubstituted or substituted by 1 to 3 methyl groups, one ethyl group, or a hydroxyl group.

Said conjugating substituent may consist of a member selected from the group consisting oiA\Z\Z\ wherein Z\ is Z2, Z2A5 or Z2A5Ag; A} and A5 are independently selected from -(CA2A3)n- , -C(Y)(CA2A3)n-, -C(Y)Y'(CA2A3)n- -C(Y)NA4(CA2A3)n- -NA4C(Y)(CA2A3)n-, -NA4(CA2A3)n, -YC(Y')(CA2A3)n-and -Y(CA2A3)n-; n = 0 - 6; Y and Y' are independently O or S; A2, A3 and A4 are independently H or C|_2 alkyl which is unsubstituted or substituted by one or more fluorines; Ag = - (C2H40)m- or -S(0)p; m = 1 - 12; p = 0 - 2; Z2 is a single bond or Z3; Z3 is selected from Z4, Z5 and Zg, wherein Z3 is unsubstituted or substituted one or more times by OH, halo, CN, N02, Ai Ajq, AgAg, NAiqA] j, C(Y)A7, C(Y)Y'A7, Y(CH2)qY'A7, Y(CH2)qA7, C(Y)NA10An, YCO^qCQONAioA! h Y(CH2)qC(Y')A9, NA10C(Y)NA10A1 b NA10C(Y)Ai h NA10C(Y)Y'A9, NA10C(Y)Z6, C(NA10)NA10A11, C(NCN)NA10An, C(NCN)SA9, NAl0C(NCN)SA9, NA10C(NCN)NA10A1 i, NA10S(O)2A9, S(0)rA9, NAioC(Y)C(Y,)NAjoAn1 NAioC(Y)C(Y')Aio orZg; q = 0, 1 or 2; r= 0-2; A7 is independently selected from H and A9; Ag is O or A9; A9 is C 1.4 alkyl which is unsubstituted or substituted by one or more fluorines; A^q is OA9 or Aj A} 1 is A7 or when A10 and A} j are as NAjqAj 1 they may together with the nitrogen form a 5 to 7 22 membered ring comprising only carbon atoms or carbon atoms and at least one heteroatom selected from 0, N and S; Z4 is C5.12 aryl or aryloxyC^alkyl; Z5 is selected from furanyl, tetrahydrofuranyl, indanyl, indenyl, tetrahydropyranyl, pyranyl, thiopyranyl, tetrahydrothiopyranyl, tetrahydrothienyl, thienyl, C3.8 cycloalkyl or C^g cycloalkyl containing one or two unsaturated bonds, and C7.1 \ polycycloalkyl; Zg is selected from N-azolyl, dioxadiazinyl, dioxadiazolyl, dioxanyl, 2-N-dioxatriazinyl, dioxazinyl, N-dioxazolyl, dioxolyl, dithiadiazinyl, dithiadiazolyl, N-dithiatriazinyl, dithiazinyl, N-dithiazolyl, 1-N-imidazolyl, N-morpholinyl, pyrollyl, tetrazolyl, thiazolyl, triazolyl, oxazinyl, oxazolyl, naphthydrinyl, oxadiazinyl, oxadiazolyl, oxatetrazinyl, oxatriazinyl, oxatriazolyl, oxazinyl, oxazolyl, pentazinyi, phthalazinyl, N-piperidinyl, N,N-piperazinyl, 1-N-pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, tetrathiazinyl, tetrazinyl, 1-N-tetrazolyl, tetroxazinyl, thiadiazinyl, thiadiazoyl, thiatetrazinyl, thiatriazinyi, thiatriazolyl, thiazolyl, triazinyl, 1-N-triazolyl, trioxadazinyl, trioxanyl, trioxazinyl, trioxazolyl, trithiadiazinyl, trithiazinyl, trithiadiazolyl; wherein Z4, Z5 or Zg may be fused to one or more other members selected independently from Z4, Z5 and Zg.

In particular embodiments of the present invention, said chromophore may comprise a chromophore having a structure set out as (x), (y) or (z) below: (x) (y) (z) ■ 23 wherein R and R' may be any of the following combinations: R 4-H 4-Me 4-Br 4-CCbMe 3,4,5-tris(OMe) 4-NCS 4-NCS 4-NCS 4-NCS 4-NCS 4-NCS R' 4-NCS 4-NCS 4-NCS 4-NCS 4-NCS 4-OMe 4-Me 4-C02Me 4-Br 4-CN 4-C02Me In another embodiment of the present invention, said chromophore may comprise a porphyrin chromophore having the structure set out below: NCS 24 wherein m = 0-6 ; p = 0-15, preferably 0-5; or the corresponding chlorin or bacteriochlorin chromophore.

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 porphyrin chromophore, a chlorin chromophore and a bacteriochlorin chromophore, each of which chromophores comprises the same porphyrin skeleton, each of which chromophores comprises one or more substituents on said porphyrin skeleton, one of which substituents is a conjugating substituent L comprising a conjugating group Z, wherein Z is a conjugating group capable of conjugating each of said chromophores to a polypeptide molecule for delivering each chromophore to one of a plurality of different specific biological targets.

Preferably, each of the other of said substituents on the skeleton is independently H or an inert substituent R which together with said conjugating substituent L and all of the other core substituents does not substantially impair the fluorescent properties of each chromophore.

It has been found that each of the chromophores in a set in accordance with the present invention, on excitation, will 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 bioconjugation to a biological target 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.

Said set of chromophores may in particular comprise two or more of a porphyrin chromophore in accordance with any aspect of the present invention, the corresponding chlorin chromophore, and the corresponding bacteriochlorin chromophore. (By "corresponding" herein is meant having the same meso-substituents around the macrocyclic core of the molecule).

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 binding 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 binding protein which is adapted to bind specifically to said biological target. Accordingly, said chromophore may be covalently linked to said binding 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 binding 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 binding protein with specificity for said specific biological target. 26 Said binding 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. In particular, said antibody may be a phage antibody, that is an antibody expressed on the surface of a bacteriophage. Alternatively said binding 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 binding 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 binding 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 27 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 binding 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 HTV 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 28 PCT/GBO1/02846 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 internalisation receptor is capable of binding said binding 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 29 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 group such as a quatenised amine or pyridyl (pyridiniumyl) group, or a phosphonium group, so as to promote the intracellular accumulation of said chromophore around the mitochondria of the cell, owing to the net negative charge on the mitochondrial membrane. This will result in the rapid and efficient killing of the cell, on production of singlet oxygen by decay of the chromophore.

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, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis or multiple sclerosis, 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, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis or multiple sclerosis, 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, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or multiple sclerosis; 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. lH/I3C 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 MALDIII 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 fim thickness). Merck Silica-Gel 60 (23.0-400 mesh) was used for flash chromatographic purification. 31 Descriptions (1) 5-(4-AcetamidophenyI)-10,15,20-tris(3,'5-dimethoxyphenyI)porphyrin 4-Acetamidobenzaldehyde (3.36 g, 0.02 mol) and 3,5-dimethoxybenzaldehyde (10 mL, 0.06 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole (5.5 mL, 0.08 mol) was added and the mixture stirred under reflux for 30 min. Upon cooling the reaction mixture was evaporated in vacuo to yield a dark purple solid. The crude mixture of porphyrin isomers was purified by flash chromatography (silica, eluent: CBhCk/EtOAc, 4:1). Relevant fractions were combined, dried (Na2S04) and evaporated in vacuo to yield 1 as a purple solid (1.55 g, 9.1%); Rf- 0.50 (silica, CtbCVEtOAc, 4:1); mp >350 °C decomp.; 'HNMR [270 MHz, CDC13] £-2.96 (2H, br s, NH), 2.23 (3H, s, NHCOCffj), 3.93 (18H s, 3, 5-OCH3), 6.99 (3H, s, 10, 15, 20-Ar-4-H), 7.07 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7.38 (6H, s, 10, 15, 20-Ar-2,6-H), 7.44 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.86-8.93 (8H, m, P-H), 10.42 (1H, br s, NtfCOCH3); l3C NMR [67.5 MHz, CDC13] £20.4, 23.9, 103.9, 113.5, 117.3, 119, 119.4, 119.6, 120, 129, 131.1, 131.3, 131.4, 131.8, 134.5, 135.6, 136.8, 139.3, 143.1, 158.6, 160.3, 167.9, 168.7; UV-vis (CH2C12) 421, 515, 551, 590, 650 nm; MS (MALDI-TOF) m/z 852 (M+, 100%). 32 (2) 5-(4-Aminophenyl)-10,15,20-tris(3,5-dimethoxyphenyI)porphyrin Porphyrin 1 (500 mg, 0.587 mmol) was dissolved in 18% HCl (100 mL) and the solution heated for 2 hours under reflux. Upon cooling the reaction mixture was evaporated in vacuo to yield a crude green solid. The solid was redissolved in a 9:1 mixture of dichloromethane/triethylamine (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL) and brine (200 mL), the organic layer separated and dried (Na2S04). Excess solvent was evaporated in vacuo and the crude purple solid purified by flash chromatography (silica, eluent: CKfeCfc/EtOAc, 4:1). Relevant fractions were combined, dried (NaaSCU) and evaporated in vacuo to yield 2 as a purple solid (426 mg, 89.7%); Rf = 0.89 (silica, CHbCVEtOAc, 4:1); mp >350 °C decomp.; >HNMR [270 MHz, CDC13] £-2.80 (2H, br s, NH), 3.96 (18H, s, 3, 5-OCH3), 6.90 (3H s, 10, 15, 20-Ar-4-H), 7.06 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7.40 (6H, s, 10, 15, 20-Ar-2,6-H), 7.98 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.93 (8H, m, f3-H); 13C NMR [67.5 MHz, CDC13] <5 20.13, 100.6, 113.9, 114.3, 115.7, 119.8, 120.1, 121.4, 130.2, 131.4, 132.7, 136.1, 144.5, 144.6, 146.5, 159.3; UV-vis (CH2CI2) Xmas 422, 517, 553, 593, 651 nm; MS (MALDI-TOF) m/z 809 (M1", 100%), 33 (3) 5-(4-Aminophenyl)-10,15,20-tris(3,5-dihydroxyphenyl)porphyrin NH2 HQ OH HO OH HO'^^'^OH To a stirred solution of 2 (1 g, 1.23 mmol) in freshly distilled chloroform (50 mL) was added boron tribromide (1.17 mL, 0.012 mol). The reaction was allowed to proceed under argon for 17 hours at room temperature. The reaction was subsequently cooled to 0 °C, water (20 mL) added and the solution stirred for a further 60 min. The reaction was evaporated to dryness in vacuo and redissolved in a 9:1 mixture of chloroform/triethylamine (500 mL). The solution was washed with water (3 x 500 mL) and brine (500 mL), the organic layer separated, dried (Na2S04), and evaporated in vacuo to yield a crude purple solid. The crude solid was purified by flash chromatography (silica, eluent: CHCl3/MeOH, 9:1). Relevant fractions were combined, dried (Na2S04) and evaporated in vacuo to yield 3 as a purple solid (667 mg, 74.5%); Rf= 0.19 (silica, CHCl3/MeOH, 9:1); mp >350 °C decomp.; *H NMR [270 MHz, (CD3)2SO] £-2.95 (2H, br s, NH), 5.56 (2H, br s, NH2), 6.69 (3H, s, 10, 15, 20-Ar-4-H), 7.02 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7..06 (6H, s, 10, 15, 20-Ar-2,6-H), 7.87 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.94 (8H, s, p-H), 9.75 (6H, br s, 3, 5-OH); 13C NMR [67.5 MHz, (CD3)2SO] 5 102.3, 112.5, 113.9, 114.1, 119.2, 119.7, 121.5, 127.5, 128.3, 130.7-131.3, 135.5, 142.8, 142.9, 148.6, 156.4, 156.5; UV-vis (CH2C12) W 422, 517, 553, 592, 649 nm; MS (MALDI-TOF) m/z 726 (M*, 100%). (4) 5-(4-AcetamidophenyI)-10,l5,20-tris(4-pyridyI)porphyrin 4-Acetamidobenzaldehyde (3.26 g, 0.02 mol) and 4-pyridinecarboxaldehyde (5.66 mL, 0.06 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole (5.4 mL, 0.08 mol) was added and the mixture stirred under reflux for 30 min. Upon cooling the reaction mixture was evaporated in vacuo to yield a dark purple solid. The crude mixture of porphyrin isomers was purified by flash chromatography (silica, eluent: CHClj/MeOH, 19:1). Relevant fractions were combined, dried (Na2SC>4) and evaporated in vacuo to yield 4 as a purple solid (526 mg, 3.9%); Rf - 0.22 (silica, CHCl3/MeOH, 19:1); mp >350 °C decomp.; 'H NMR [270 MHz, CDC13] 5 -2.79 (2H, br s, NH), 2.49 (3H, s, NHCOCtf;), 8.07 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 8.21-8.28 (8H, m (overlapping), 5-Ar-2,6-H & 10, 15, 20-Py-2,6-H), 8.84-9.06 (8H, m, (3-H), 9.10-9.15 (6H, m, 10, 15, 20-Py-3,5-H), 10.35 (1H br s, NtfCOCH3); 13C NMR [67.5 MHz, CDCI3] £26.8, 106.9, 110.1, 110.2, 117.9, 121.1, 121.5, 122.1, 122.2, 123.3, 123.8, 123.9, 134.7, 140.1, 142.5, 145.1, 148.2, 149, 149.3, 149.4, 149.6, 150.1, 175.2; UV-vis (CH2C12) ?w418, 514, 548, 587, 644 nm; MS (MALDI-TOF) m/z 675 (M\ 100%). (5) 5-(4-Aminophenyl)-10,15,20-tris(4-pyridyl)porphyrin Porphyrin 4 (500 mg, 0.74 mmol) was dissolved in 18% HCI (100 mL) and the solution heated for 2 hours under reflux. Upon cooling the reaction mixture was evaporated in vacuo to yield a crude green solid. The solid was redissolved in a 9:1 mixture of dichloromethane/triethylamine (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL) and brine (200 mL), the organic layer separated and dried (Na2S04). Excess solvent was evaporated in vacuo and the purple crude solid purified by flash chromatography (silica, eluent: CHC^/MeOH, 20:1). Relevant fractions were combined, dried (Na2S04) and evaporated in vacuo to yield 5 as a purple solid (422 mg, 90.1%); Rf= 0.31 (silica, CHCl3/MeOH, 20:1); mp >350 °C decomp.; lHNMR [270 MHz, CDCI3] £-2.86 (2H, br s, NH), 4.09 (2H, br s, NHa), 7.08 (2H, m, ./* = 8 Hz, 5-Ar-3,5-H), 7.98 (2H, m, ./* = 8 Hz, 5-Ar-2,6-H), 8.16 (6H, m, J* = 5 Hz, 10, 15, 20-Py-2,6-H), 8.80-9.01 (8H, m, (3-H), 9.04 (6H, m, J* = 5 Hz, 10, 15, 20-Py-3,5-H); 13C NMR [67.5 MHz, CDCI3] 8 113.6, 116.7, 117.4, 117.9, 122.7, 129.5, 131.7, 135.9, 146.5, 148.4, 148.5, 149.8, 150.2; UV-vis (CH2C12) 418, 515, 552, 592, 653 nm; MS (FAB) m/z 633 (M+, 100%). 36 (6) 5-(4-Acetamidophenyl)-10,15,20-tris(3-pyridyl)porphyrin HN Me 4-Acetamidobenzaldehyde (5 g, 0.031 mol) and 3-pyridinecarboxaldehyde (8.67 mL, 0.092 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole (8.5 mL, 0.123 mol) was added and the mixture stirred under reflux for 30 min. Upon cooling the reaction mixture was evaporated in vacuo to yield a dark purple solid. The crude mixture of porphyrin isomers was purified by flash chromatography (silica, eluent: CHCb/MeOH, 19:1). Relevant fractions were combined, dried (Na2SC>4) and evaporated in vacuo to yield 6 as a purple solid (0.96 g, 4.6%); Rf= 0.26 (silica, CHCU/MeOH, 19:1); mp >350 °C decomp.; *HNMR [270 MHz, CDC13] <5-2.97 (2H, br s, NH), 2.17 (3H, s, NHCOCtfj), 7.40 (2H, m, ./* = 8 Hz, 5-Ar-3,5-H), 7.49 (3H, m, 10, 15, 20-Py-5-H), 7.98 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.21-8.33 (3H, m, 10, 15, 20-Py-6-H), 8.57-8.82 (11H, m (overlapping), 10, 15, 20-Py-4-H & p-H), 8.99 (1H, br s, NffCOCH3), 9.26 (3H, m, 10, 15, 20-Py-2-H); UV-vis (CH2C12) ?wx419, 516, 552, 592, 648 nm; MS (MALDI-TOF) w: z 675 (M+, 100%). 37 (7) 5-(4-Aminophenyl)-10,15,20-tris(3-pyridyl)porphyrin Porphyrin 6 (300 mg, 0.45 mmol) was dissolved in 18% HC1 (100 mL) and the solution heated for 2 hours under reflux. Upon cooling the reaction mixture was evaporated in vacuo to yield a crude green solid. The solid was redissolved in a 9:1 mixture of dichloromethane/triethylamine (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL) and brine (200 mL), the organic layer separated and dried (Na2SO.i). Excess solvent was evaporated in vacuo and the purple crude solid purified by flash chromatography (silica, eluent: CHCl3/MeOH, 20:1). Relevant fractions were combined, dried (Na^SOj) and evaporated in vacuo to yield 7 as a purple solid (206 mg, 68.5%); Rf= 0.38 (silica, CHCl3/MeOH, 20:1); mp >350 °C decomp.; *HNMR [270 MHz, CDCl3] 8-2.74 (2H, br s, NH), 3.93 (2H, br s, NHa), 6.91 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7.67 (3H, m, 10, 15, 20-Py-5-H), 7.93 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.48 (3H, m, 10, 15, 20-Py-6-H), 8.79-9.02 (11H, m (overlapping), 10, 15, 20-Py-4-H & (J-H), 9.47 (3H, s, 10, 15, 20-Py-2-H); 13C NMR [67.5 MHz, CDC13] 8 113.3, 115.6, 116.1, 116.7, 121.9, 122.3, 131, 131.4, 131.8, 132.1, 132.3, 135.7, 137.5, 137.7, 137.8, 140.8, 146.3, 149, 149.2, 153.5; UV-vis (CH2C12) ^ax420, 517, 553, 597, 649 nm; MS (MALDI-TOF) m/z 632 (M+, 100%). 38 PCT/GBO1/02846 (8) 5-(4-Acetamidophenyl)-15-(4-methoxyphenyl)porphyrin, The DDP was synthesised according to the method of Dolphin et al.( 1998 5-Phenyldipyrromethane and 5, 15-Diphenylporphyrin Org. Synth. 76, 287-293 incorporated herein by reference) The resulting mixture of three porphyrins was chromatographed, eluting initially with DCM to allow removal of 5,15-(4-methoxy)-DPP, and then continuing with ethyl acetate/DCM (1 ;4) to elute the required product as purple crystals (150 mg, 12%); Rf = 0.40 (DCM/MeOH, 19:1); mp 305-307°C (decomposed); UV-vis (DCM) ^max (relative intensity) 410 (1.0), 502 (0.04), 538 (0.02), 578 (0.015), 630 (0.01) nm; *H NMR (270 MHz, CDC13) 5 10.35 (s, 2H, 10+20-H), 9.43 (d, 4H, J = 4.8 Hz, /?-#), 9.14 (d, 4H, J = 4.8 Hz, 0-H), 8.65 (m, 2H, J = 7.2 Hz, 5-m-Ar\ 8.22-8.12 (m, 4H, (overlapping), J = 8.1 Hz, 5+15-o-Ar), 7.56 (m, 2H, J = 8.1 Hz, 15-m-i4r), 4.14 (s, 3H, CHS), -3.00 (br s, 2H, NH); MALDI-MS m/z 550.3 (Tvf, 100%). (9) 5-(4-Aminophenyl)-15-(4-methoxyphenyI)porphyrin, -(4-Acetamido phenyl)-15-(4-methoxyphenyl)porphyrin 8 (1 eq., 100 mg, 0.182 mmol) was dissolved in 5 M aqueous HCl (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 ofDCM/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 (Na^SCU), then concentrated in vacuo. The crude purple solid was chromatographed, eluting with DCM, and gave the desired porphyrin as a purple crystalline solid (51 mg, 54%), Rf= 0.30 (DCM), mp 300°C (decomposed); UV-vis (DCM) (relative intensity) 410 (1.0), 503 (0.045), 538(0.02), 578 (0.015), 630 (0.005) nm; Fluorescence (DCM) ?Wx 634 nm {X excitation = 410 nm); 'H NMR (270 MHz, CDC13) 5 10.30 (s, 2H, 10+20-H), 9.39 (d, 4H, J = 4.9 Hz, JS-H), 9.17 (d, 2H, J = 4.9 Hz, fi-H), 9.10 (d, 2H, J = 4.9 Hz, 0-H), 8.19 (m, 2H, J = 8.8 Hz, 15-o-Ar\ 8.07 (m, 2H, J = 8.1 Hz, 5-o~Ar), 7.35 (m, 2H, J = 8.8 Hz, I5-m-Ar\ 7.14 (m, 2H, J.= 8.1 Hz, 5-m-Ar), 4.13 (s, 3H, CHS), 4.08 (br s, 2H, NH), -3.06 (br s, 2H NH)\ MALDI-MS m/z 508.3 ([M+l]+, 100%). ES-HRMS calcd. for C33H26N50 ([M+lf) 508.2137, found 508.2144. 39 PCT/GBO1/02846 (10) 17,18-Dihydroxy-5-(4-aminophenyl)-15-(4-methoxyphenyl)chlorin and (11) 7,8-dihydroxy 5-(4-aminophenyl)-15-(4-methoxyphenyI)chlorin regioisomers, Porphyrin 9 (28 mg, 55.2 pjnol) was converted into the required mixture of chlorin regioisomers following 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 andPhthalocycinines 4, 655-658) The crude reaction mixture was then chromatographed, eluting with 1% MeOH in DCM. First, some un-reacted starting material was eluted, then the higher Rf chlorin isomer 10 as a brown-purple crystalline solid; Rf~, 0.28 (DCM/MeOH, 19:1). The lower Rr isomer 11 was obtained by further elution with 2.5% MeOH in DCM and gave also a brown-purple crystalline solid (fy= 0.17 (DCM/MeOH, 19:1).

High Rf chlorin regioisomer (17,18-dihydroxy-15-(4-methoxy phenyl)-5-(4-aminophenyl)chlorin assigned previously^#),) (7.0 mg, 24%), mp 165-167°C (decomposed); UV-vis (DCM) Xmax (relative intensity) 401 (0.99), 414 (1.0), 503 (0.08), 535 (0.07), 582 (0.035), 636 (0.22) nm; Fluorescence (DCM) Xmax 639 nm (X excitation = 412 nm); lH NMR (270 MHz, 10% CD3OD in CDC13) 5 9.95 (s, 1H, 10-H), 9.42 (s, 1H, 20-H), 9.17 (d, 1H, J = 4.8 Hz, J3-H), 9.03 (d, 1H, J = 4.0 Hz, p~H), 8.97 (s, 2H, J3-H) 8.78 (d, 1H, J = 4.8 Hz, jB-H), 8.51 (d, 1H, J = 4.8 Hz, P-H), 8.05 (m, 2H, J = 8.9 Hz, o-Ar), 7.94 (m, 2H, J = 8.1 Hz, o '-Ar), 7.25 (m, 2H, J = 8.9 Hz, m-Ar), 7.12 (m, 2H, J = 8.1 Hz, m '-Ar), 6.42 (d, 1H, J = 6.5 Hz, 17-H), 6.03 (d, 1H, J = 6.5 Hz, I8-H), 4.08 (s, 3H., CHs), (NH's exchanged & OH's not observed); MALDI-MS m/z 542.2 ([M+H]+, 100%); ES-HRMS calcd. for C33H28N5O3 ([M+Hf) 542.2192, found 542.2187.

Low Rf chlorin regioisomer (7,8-dihydroxy-5-(4-aminophenyl)-15-(4-methoxyphenyl)chlorin) (8.5 mg, 30%), mp 168-171°C (decomposed); UV-vis (DCM) ^ (relative intensity) 401(0.99), 413 (1.0), 507 (0.08), 536 (0.06), 586 (0.025), 637 (0.20) nm; Fluorescence (DCM) Amax 639 nm (A. excitation = 412 nm); lH NMR (270 MHz, 10% CD3OD in CDCI3) 5 9.96 (s, 1H, 20-H), 9.40 (s, 1H, 10-H), 9.18 (d, 1H, J = 4.8 Hz, p-H), 9.05 (d, 1H, J = 4.8 Hz, P-H), 8.98 (d, 1H, J = 4.0 Hz, p-H) 8.92 (d, 1H, J = 4.0 Hz, p-H), 8.74 (d, 1H, J = 4.0 Hz, p-H), 8.58 (d, 1H, J = 4.0 Hz, p-H), 8.13 (m, 1H, J = 8.9 Hz, o-Ar), 8.08 (m, 1H, J= 8.9 Hz, o-Ar), 7.95 (m, 1H, J = 8.1 Hz, 0'-Ar), 7.79 40 (m, 1H. J = 8.1 Hz, o -Ar), 7.36 (m, 1H, J = 8.9 Hz, m-Ar), 7.30 (m, 1H, J = 8.9 Hz, m-Ar), 7.11 (m, 1H, J = 8.1 Hz, m '-Ar), 7.05 (m, 1H, J = 8.1 Hz, m -Ar), 6.42 (d, 1H, J = 6.5 Hz, 7-H), 6.09 (d, 1H, J = 6.5 Hz, 8-H), 4.11 (s, 3H, CH3), (NH's exchanged & OH's not observed); MALDI-MS m/z 542.2 ([M+H]+, 100%) ES-HRMS calcd. for C33H2gN503 ([M+H]") 542.2192, found 542.2185. (12) 5-(4-Fluorenylmethylaminophenyl)-15-(4-methoxyphenyl)porphyrin, To a stirred solution of porphyrin 9 (28 mg, 55 p.mol) 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 (Na2S04), filtered and concentrated in vacuo. The required porphyrin was obtained by chromatography, eluting with DCM. The desired porphyrin was obtained as purple crystals (38 mg, 95%), R/= 0.39 (DCM), mp 292-295°C (decomposed); UV-vis (DCM) Amax (relative intensity) 410 (1.0), 505 (0.042), 541 (0.02), 578 (0.015), 633 (0.01) nm; Fluorescence (DCM) 635 nm (X excitation = 410 nm); *H NMR (270 MHz, CDCI3) 5 10.35 (s, 2H, 10+20-H), 9.69 (br. s, 1H, NH), 9.44 (d, 4H, J = 4.8 Hz, P-H), 9.12 (d, 4H, J = 4.8 Hz, p-H), 8.20-8.17 (m (overlapping), 4H, J = 8.1 Hz, 5+15-o-Ar), 7.85 (m, 4H, 5+15-m-Ar), 7.76-7.66 (m, m,fluoreno-Ar), 7.51-7.30 (m, 6H,jluoreno-Ar), 4.69 (d, 2H, J = 7.2 Hz, CH2), 4.30 (t, 1H, J = 7.2 Hz, CH), 4.13 (s, 3H, CHS), -3.15 (br. s, 2H, NH)] MALD-MS m/z 731.5 ([M+H]+, 100%), 508.3 ([M-FMOC+2f, 50%); ES-HRMS calcd. for C48H36N5O3 ([M+H]+) 730.2818, found 730.2809. (13, 14) cis/frflns-7,8,17,18-Tetrahydroxy-5-(4-fluorenylmethylaminophenyl)-15-(4-methoxyphenyl) bacteriochlorins Porphyrin 12 (35 mg, 48.0 fimol) was converted into the required mixture of 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 41 andPhthalocyanin.es 4, 655-658 - incorporated herein by reference) (reaction carried out using 1,4-dioxane (5 mL) to allow dissolution of 12). The crude reaction mixture was chromatographed, eluting initially with 1% MeOH in DCM to remove chlorin byproducts. Further elution with 2% MeOH/DCM allowed isolation of both stereoisomeric bacteriochlorin tetrols. The higher Rr trans bacteriochlorin isomer 13 was isolated as a pink-green crystalline solid, (6 mg, 15%), Rf= 0.25 (DCM/MeOH, 19:1), mp 142-145°C (decomposed); UV-vis (DCM) (relative intensity) 374 (1.0) 512 (0.23), 702 (0.52) nm; Fluorescence (DCM) A.max 708 nm (X excitation = 512 nm); LH NMR (270 MHz, 10% CD3OD in CDCI3) 6 9.20 (s, 2H, 10+20-H), 8.78 (d, 2H, J = 4.0 Hz, p-H), 8.36 (d, 2H, J = 4.0 Hz, P-H), 7.95 (m, 2H, o-Ar), 7.85 (m, 2H, J = 7.3 Bz,fluoreno-Ar), 7.79 (m, 2H o '-Ar), 7.65 (m. 2H, m -Ar), 7.47-7.38 (m, 6H, fluoreno-Ar), 7.24 (m, 2H, m-Ar), 6.27-6.24 (m, 2H, 7+17-H), 5.85 (m, 2H, 8+18-H), 4.65 (d, 2H, J = 7.2 Hz, CH2), 4.39 (t, 1H, J = 7.2 Hz, CH), 4.06 (s, 3H, CH3), -1.94 (br s (partly exchanged), 2H, NH), (OH's not observed); MALDI-MS m/z 800.4 ([M+H]+, 100%); ES-HRMS calcd. for C4gBUoNj07 ([M+H]+) 798.2927, found 798.2921.

The lower Rf cw-bacteriochlorin isomer 14 was isolated as a pink-green crystalline solid, (8.5 mg, 21%), Rf= 0.2 (DCM/MeOH, 19:1), mp 148-150°C (decomposed); UV-vis (DCM) \max (relative intensity) 374 (1.0) 512 (0.24), 703 (0.54) nm; Fluorescence (DCM) Xmas 708 nm (X excitation = 512 nm); lH NMR (270 MHz, 10% CD3OD in CDCI3) 5 9.12 (s, 2H, 10+20-H), 8.76 (d, 2H, J = 4.8 Hz, P-H), 8.34 (d (overlapping), 2H, J = 4.8 Hz, P~H), 8.02 (m, 2H, o-Ar), 7.85 (m (obscured), 2H, J = 8.0 Hz, o -Ar), 7.83 (m, 2H, J = 7.3 Yh., fluoreno-Ar), 1.16 (m, 2H, J = 8.0 Hz, m '-Ar\ 7.50-7.38 (m, 6H, fluoreno-Ar), 7.24 (m, 2H, m-Ar), 6.27-6.23 (m, 2H, 7+ 17-H), 5.85-5.82 (m, 2H, 8+18-H), 4.65 (d, 2H, J = 7.2 Hz, CH2), 4.39 (t, 1H, J = 7.2 Hz, CH), 4.05 (s, 3H, Cffj), -1.88 (br. s (partly exchanged), 2H, NH), (OH's not observed); MALDI-MS m/z 800.4 ([M+Hf, 100%); ES-HRMS calcd. for C48H4oN507 ([M+H]+) 798.2927, found 798.2921. (15) 5-(3,4,5-Trismethoxyphenyl)dipyrromethane 3,4,5-Trismethoxybenzaldehyde (5.0 g, 25.5 mmol) was dissolved in freshly distilled pyrrole (75 ml) and the solution degassed by bubbling with dry N2 for 10 min. TFA (0.075 eq. , 0.15 ml, 1.91 mmol) was added and the mixture stirred under N2 until no starting aldehyde could be detected by TLC (ca. 10 min). The reaction mixture was 42 concentrated in vacuo at water aspirator pressure (evaporator water bath temp 75°C ) then under high vacuum for 16 h to remove excess pyrrole. The crude product was recrystallised from hot ethylacetate/ nHexane and afforded the required dipyrromethane as a white solid, ( 5.41 g, 68%): vmax (nujol mull)/ cm"1 3378 (br. NH), 1594 (C=C), 1233, 1040; UV-VIS (MeOH) Xmax/ (rel. intensity) 222 (1.0), 280 (0.75) nm; 5H(270 MHz; CDC13) 8.07 (2H, br. s, NH), 7.53 (2H, m, 1-H), 6.68 (2H, s, 2'-H), 6.37 (2H, m, 2-H), 5.93 (2H, m, 3-H), 5.38 (1H, s, methane), 3.80 (3H, s, 4'-OCH3), 3.73 (6H, s, 3^5'-OCH3y, 5C(68 MHz; CDCI3) 152.7 (CH, 2'-C), 137.3 (q, 3'+5'-C), 136.1 (q, 4'-C), 13 1.8 (q, 4-C), 116.7 (CH, 1-C), 107.9 (CH, 2-C), 106.6 (CH, 3-C), 104.9 (q, l'-C), 60.3 (Cffj), 55.5 (CHS), 43.7 (CH,, methane); MS (MALDI) m/e 311.2 (100%, (M-l)+). (16) 5-(4-AcetomidophenyI)dipyrromethane The dipyrromethane was synthesised using the general procedure detailed above using the same molar quantity of starting aldehyde. The crude reaction mixture was chromatographed on flash silica-gel (350 ml), (dry loaded on to 50 ml flash silica-gel from ethylacetate) and eiuted with 40% ethylacetate/ DCM and afforded the pure product as an off white solid, (4.3 g, 50%): vmax (nujol mull)/ cm"1 3409 (NH, amide), 3248 (br. NH), 1650 (C=0), 1593 (C=C), 1320, 1009; UV-VIS (MeOH) W/(rel. intensity) 224 (1.0) nm; 5H(270 MHz; CDCI3) 8.00 (2H, br. s, NH), 7.40 (2H, d, J = 8.5 Hz, o-Ar ), 7.30 (1H, br. s, NH-acetomido), 7.13 (2H, d, J = 8.5 Hz, m-Ar), 6.68 (2H, m, 1-H), 6.16 (2H, m, 2-H), 5.90 (2H, m, 3-H), 5.42 (1H, s, methane), 2.14 (3H, s, NHCMA 5C(68 MHz; CDCI3) 168.4 (q, COCHs), 138.2 (q, 4'-C), 136.5 (q, 2'-C), 132.4 (q, 4-C), 128.9 (CH, 2'-C), 120.3 (CH, 3'-C), 117.2 (CH, 1-C), 108.4 (CH, 2-C), 107.1 (CH, 3-C), 43.4 (CH., methane), 24.5 (CH3); MS (MALDI) m/e 279.4 (100%, (M)+). (17) 5-(4-Methoxyphenyl)dipyrromethane The dipyrromethane was synthesised using the general procedure detailed above using the same molar quantity of starting aldehyde. The crude reaction mixture was chromatographed on flash silica-gel (350 ml), (dry loaded on to 50 ml flash silica-gel from ethylacetate) and eiuted with 30% nHexane/ DCM and afforded the pure product as an off white solid, (4.3 g, 50%): vmax (nujol mull)/ cm'1, 3382 (br. NH), 1598 (C=C), 1300, 1050; UV-VIS (MeOH) Xmax/ (rel. intensity) 224 (1.0) nm; SH(270 MHz; CDCI3) 43 7.87 (2H, br. s, NH), 7.10 (2H, d, J = 8.8 Hz, m-Ar ), 6.83 (2H, d, J = 8.8 Hz, o-Ar), 6.66 (2H, m, 1-H), 6.14 (2H, m, 2-H), 5.89 (2H, m, 3-H), 5.40 (1H, s, methane)-, MS (MALDI) m/e 252.4 (100%, (M)+).

Example 1 -(4-Isothiocyanatophenyl)-10,15,20-tris(3,5-dihydroxyphenyl)porphyrin To a stirred solution of 3 (100 mg, 0.137 mmol) in freshly distilled THF (25 mL) was added 1, r-thiocarbonyldi-2(l//)-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 purple solid. The solid was dissolved in a minimal amount of chloroform/methanol (9:1) and purified by flash chromatography (silica, eluent. CHCl3/MeOH, 9:1). Relevant fractions were combined, dried (Na^SCU) and evaporated in vacuo to yield the above compound as a purple solid (67.5 mg, 63.8%); Rf = 0.29 (silica, CHCl3/MeOH, 9:1); mp >350 °C decomp.; *H NMR [270 MHz, CDCI3/CD3OD, 3:1] 56.77 (3H, s, 10, 15, 20-Ar-4-H), 7.12 (6H, s, 10, 15, 20-Ar-2,6-H), 7.64 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 8.19 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.76-9.0 (8H, m, p-H); 13C NMR [67.5 MHz, CDCI3/CD3OD, 3:1] £101.9, 107.1, 114.7, 117.6, 119.9, 120, 120.1, 123.9, 130.9, 134, 135.2, 136.1, 141.2, 142, 143.6, 155.8; UV-vis (MeOH) 44 ^ma\-422, 516, 552, 592, 648 nm; HRMS (ES) mlz calc'd for C45H29N5O6S [M+H]+ 768.1914, found 768.1908.

Example 2 -(4-IsothiocyanatophenyI)-l0,15,20-tris(4-pyridyI)porphyrin To a stirred solution of 5 (100 mg, 0.158 mmol) in freshly distilled dichloromethane (20 mL) was added l,r-thiocarbonyldi-2(l/f)-pyridone (320 mg, 1.38 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 purple solid. The solid was dissolved in a minimal amount of chloroform and purified by flash chromatography (silica, eluent: CHCl3/MeOH, 49:1). Relevant fractions were combined, dried (Na2S04) and evaporated in vacuo to yield the above compound as a purple solid (104 mg, 97.5%); 0.57 (silica, CHCl3/MeOH, 49:1); mp >350 °C decomp.; *11 NMR [270 MHz, CDCI3] £-2.91 (2H, br s, NH), 7.65 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 8.15-8.21 (8H, m (overlapping), 10, 15, 20-Py-2,6-H & 5-Ar-2,6-H), 8.67 (8H, br s, (3-H), 9.06 (6H, m, J* = 5 Hz, 10, 15, 20-Py-3,5-H); 13C NMR [67.5 MHz, CDC13] S117.4, 117.6, 119.7, 124.7, 129.3, 131.6, 135.4, 136.9, 140.6, 148.4, 149.8; UV-vis (CH2C12) Vax 417, 514, 548, 587, 643 nm; HRMS (ES) m/z calc'd for C42H26N8S (M+H) 675.2079, found 675.2078. 45 -■ Example 3 -(4-Isothiocyanatophenyl)-10,15,20-tris(3-pyridyl)porphyrin (160) NCS To a stirred solution of 7 (200 mg, 0.316 mmol) in freshly distilled dichloromethane (40 mL) was added l,r-thiocarbonyldi-2(l£0-pyridone (640 mg, 2.76 mmol). The reaction was allowed to proceed under argon for 17 hours at room temperature. Excess solvent was evaporated in vacuo to yield a crude purple solid. The solid was dissolved in a minimal amount of chloroform and purified by flash chromatography (silica, eluent: CHCb/MeOH, 49:1). Relevant fractions were combined, dried (NaiSCU) and evaporated in vacuo to yield the above compound as a purple solid (171 mg, 80.3%); Rf= 0.55 (silica, CHCl3/MeOH, 49:1); mp >350 °C decomp.; ^NMR [270 MHz, CDCI3] £-2.83 (2H, br s, NH), 7.65 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7.78 (3H, m, 10, 15, 20-Py-5-H), 8.20 (2H, m, J*= 8 Hz, 5-Ar-2,6-H), 8.54 (3H, m, 10, 15, 20-Py-6-H), 8.83-8.88 (8H, m, (3-H), 9.07 (3H, m, 10, 15, 20-Py-4-H), 9.07 (3H, s, 10, 15, 20-Py-2-H); 13C NMR [67.5 MHz, CDC13] £116.6, 122.1, 124.2, 131.5, 135.5, 137.7, 140.8, 140.9, 149.2, 153.5; UV-vis (CH2C12) W421, 513, 547, 587, 657 nm; MS (MALDI-TOF) m/z 674 (M+, 100%). 46 PCT/GBO1/02846 Example 4 -(4-Isothiocyanatophenyl)-10,15,20-tris(4-yV-methyIpyridiniumyI) porphyrin triiodide NCS Me To a solution of Example 2 (50 mg, 0.074 mmol) in anhydrous DMF (10 mL, distilled from CaH2, 0.1 torr) was added iodomethane (1 mL, 0.016 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 torr) at 30-40 °C to yield the above compound as a lustrous purple solid (77 mg, 95%); Rf = 0.32 (silica, H20/sat.aq. KN03/MeCN, 1:1:8); mp >350 °C decomp.; LH NMR [270 MHz, (CD3)2SO] <5-3.03 (2H, br s, NH), 4.74 (9H, br s, jV-CH3-pyridine), 7.96 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 8.32 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 9.03 (6H, m,J* = 6 Hz, 10, 15, 20-Py-2,6-H), 9.16 (8H, m, p-H), 9.50 (6H, m, J* = 6 Hz, 10, 15, 20-Py-3,5-H); 13C NMR [67.5 MHz, (CD3)2SO] <547.9, 114.7, 115.3, 121.1, 124.8, 130.6, 132, 134.7, 135.4, 139.7, 144.1, 156.3, 156.4; UV-vis (H20) ^423,520, 585 nm;MS (FAB) m/z 719 (M~, 100%), 704 (M-CH3, 26%), 689 (M-2CH3, 20%), 674 (M-3CH3, 5%); HRMS (ES) mlz calc'd for C45H35N8S (M+H) 719.2705, found 719.2686. 47 Example 5 -(4-Isothiocyanatophenyl)-10,15,20-tris(3-Ar-methylpyridiniumyI) porphyrin triiodide NCS To a solution ofExample 3 (50 mg, 0.074 mmol) in anhydrous DMF (5 mL, distilled from CaH, 0.1 torr) was added iodomethane (1 mL, 0.016 mol). The reaction was stirred under argon for 4 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 torr) at 30-40 "C to yield the above compound as a lustrous purple solid (72 mg, 89%); Rf = 0.46 (silica, H20/sat.aq. KN03/MeCN, 1:1:8); mp >350 °C decomp.; *H NMR [270 MHz, (CD3)2SO] <5-3.07 (2H, br s, NH), 4.69 (9H, br s, jY-CH3-pyridme), 7.97 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 8.31 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.64 (3H, m, 10, 15, 20-Py-5-H), 9.03-9.25 (8H, m, P-H), 9.35 (3H, m, 10, 15, 20-Py-6-H), 9.57 (3H, m, 10, 15, 20-Py-4-H), 10.03 (3H, s, 10, 15, 20-Py-2-H); 13C NMR [67.5 MHz, (CD3)2SO] £48.3, 112.3, 112.9, 120.7, 124.8, 126.3, 126.4, 126.6, 130.6, 132.1, 132.3, 132.4, 132.6, 132.8, 133.1, 133.4, 134.7, 135.4, 139.8, 139.9, 140, 145.5, 145.6, 147.4, 147.5, 147.8, 147.9, 148.5, 155.9; UV-vis (H20) Amax419, 516, 552, 581\ 637 nm; MS (MALDI-TOF) m/z 689 ([M-2CH3f, 100%). 48 Example 6 -(4-IsothiocyanatophenyI)-10,15,20-tris(4-Ar-methylpyridiniuinyl) porphyrin trichloride Me Cf To a solution of Example 4 (30 mg, 0,027 mmol) in anhydrous methanol (30 mL) was added Amberlite® IRA 400 (lg) and the mixture stirred for 1 hour at room temperature. Amberlite® IRA 400 resin was filtered under vacuum and the porphyrin filtrate recovered, dried (Na^SO^ and evaporated in vacuo to yield the above compound as a water soluble purple solid (22 mg, 96.4%). Porphyrins of Examples 4 and 6 were distinguished only by their respective solubility in water.

Example 7 -(4-Isothiocyanatophenyl)-10,15,20-tris(4-jV-methylpyridiniumyl) porphyrin trichloride 49 -- NCS CI Me Me CI CI To a solution of 5 (30 mg, 0.027 mmol) in anhydrous methanol (30 mL) was added Amberlite® IRA 400 (lg) and the mixture stirred for 1 hour at room temperature. Amberlite® IRA 400 resin was filtered under vacuum and the porphyrin filtrate recovered, dried (Na2S04) and evaporated in vacuo to yield the above compound as a water soluble purple solid (21 mg, 92.0%). Porphyrins of Examples 5 and 7 were distinguished only by their respective solubility in water.

Example 8 17,18-Dihydroxy-5-(4-isothiocyanatophenyl)-15-(4-methoxyphenyl)chlorin, The higher Rfregioisomeric chlorin 10 (17.5 mg, 23.2 |o.mol) was converted into the corresponding isothiocyanate according to the following method. To a stirred solution of 10 (1 eq., 50 mg, 0.099 mmol) in freshly distilled DCM (20 mL)was added 1,1'-thiocarbonyldi-2(li/)-pyridone (2 eq., 46 mg, 0.198 mmol). The reaction was allowed to stir under argon for 2 h at room temperature, after which the reaction mixture was filtered, and concentrated then chromatographed, eluting with 1% MeOH in DCM to afford the title compound. The title compound was isolated as a brown-purple crystalline solid, (17 mg, 90%), Rf= 0.36 (DCM/MeOH, 19:1), mp 155-158°C (decomposed); UV-vis (DCM) Anux (relative intensity) 410 (1.0) 505 (0.09), 534 (0.06), 586 (0.04), 637 (0.18) nm; Fluorescence (DCM) "Km^ 639 nm (A. excitation = 412 nm); ^HNVCR. (270 MHz, 10% CD3OD in CDCI3) 5 10.0 (s, 1H, I0-H), 9.45 (s, 1H, 20-H), 9.20 (d, 1H, J = 50 4.8 Hz, P-H), 9.06 (d, 1H, J = 4.0 Hz, p-H), 9.02 (d, 1H, J = 4.8 Hz, p-H) 8.84 (d, 1H, J = 4.8 Hz, P-H), 8.64 (d, 1H, J = 4.0 Hz, p-H), 8.55 (d, 1H, J = 4.8 Hz, P-H), 8.21 (m, 1H, J = 8.1 Hz, 5-o-Ar), 8.15 (m, 1H, J = 8.1 Hz, 5-o-Ar), 8.05 (m, 1H, J = 8.9 Hz, 15-o-Ar) 7.93 (m, 1H, J = 8.9 Hz, J 5-o-Ar), 7.65 (m, 2H, 5-m-Ar), 7.24 (m, 2H, 15-m-Ar), 6.43 (d, 1H, J = 6.5 Hz, 17-H), 6.04 (d, 1H, J = 6.5 Hz, 18-H), 4.08 (s, 3H, CH3), (NH's exchanged & OH's not observed); MALDI-MS m/z 583.7 ([M=H]+, 100%); ES-HRMS calcd. for C34H36N5O3S ([M+Hf) 584.1757, found 584.1756.

Example 9 cis-7,8,17,18-Tetrahydroxy-5-(4-isothiocyanatophenyl)-15-(4-methoxyphenyl)bacteriochIorin The cz.s-bacteriochlorin 14 (8.5 mg, 10.7 |j.mol) in 25%i MeOH in DCM (1.25 mL) was treated with piperidine (50 eq., 53 p.1, 0.53 mmol) and left to stir for a period of 3 h at room temperature under N2 with light excluded. The reaction mixture was concentrated in vacuo (0.1 torr) to remove all traces of piperidine. The crude amine was then converted into the required isothiocyanate following the procedure described above The cis-bacteriochlorirusothiocyanate was isolated as a pink-green crystalline solid (5.0 mg, 76%), Rf= 0.40 (DCM/MeOH, 19:1), mp 132-135°C (decomposed); UV-vis (DCM) >,max (relative intensity) 375 (1.0) 516 (0.22), 702 (0.48) nm; Fluorescence Xmax 709 nm (A, excitation =516 nm); ^NMR (270 MHz, 10% CD3OD in CDCI3) 5 9.20 (s, 1H, meso-H), 9.18 (s, 1H, meso-H), 8.77 (d, 2H, J = 4.8 Hz, P-H), 8.40 (d, 1H, J = 4.8 Hz, p-H), 8.34 (d, 1H, J = 4.8 Hz, P-H), 8.14 (m, 2H, o-Ar), 8.05 (m, 2H, o'-Ar), 7.42-7.08 (m, 4H, 5+15-m-Ar), 6.20 (m, 2H, 7+17-H), 5.98 (m, 1H, 8-H), 5.93 (m, 1H, 18-H), 4.04 (s, 3H, C-Hs), -1.80 (br s, 2H, partly exchanged-A®), (OH's not observed); MALDI-MS m/z 618.9 ([M+H]+, 100%); ES-HRMS calcd. for Ca^sNjOjS ([M+H]+) 618.1815, found 618.1810.

Examples 10, 11, 12, 13 17,18-Dihydroxy-5-(4-acetamidophenyl)-15-(4-methoxyphenyl)chIorin/7,8-dihydroxy-5-(4-acetamidophenyl)-15-(4-methoxyphenyl)chlorin regioisomers, and 51 PCT/GBO1/02846 c'i.s/fra/w-7,8,17,18-tetrahydroxy-5-(4-acetamidophenyl)-15-(4-methoxypheny))bacteriochlorin stereoisomers, Porphyrin 8 (100 mg, 0.18 mmol) was converted, in a single reaction, to a mixture of chlorin diols/bacteriochlorin tetrols following the procedure of Sutton et al. After 38 h the reaction was stopped. The crude reaction mixture was then chromatographed, eluting with 2% MeOH in DCM to give first, some un-reacted starting material then the higher Rr chlorin isomer of Example 10 as a brown-purple crystalline solid (5 mg, 5%). The lower Rf isomer of Example 11 was obtained by further elution with 3.5% MeOH in DCM and gave also a brown-purple crystalline solid (7.0 mg, 7%). Further elution with 5% MeOH in DCM afforded the required frara/m-bacteriochlorin tetrols of Examples 12 and 13 respectively as pink/green solids (5.0 mg, 5%) and (7.0 mg, 7%) respectively.

High Rf chlorin regioisomer of Example 10 (17,18-dihydroxy-15-(4-methoxyphenyl)-5-(4-acetamidophenyl) chlorin assigned on the basis of past data)(26) Rf= 0.40 (DCM/MeOH, 37:3), mp 186-188°C (decomposed); UV-vis (DCM) ^max (relative intensity) 410(1.0), 505.5 (0.12), 535 (0.08), 585.5 (0.05), 637 (0.18) nm; Fluorescence (DCM) Ami>x 639 nm (X excitation = 410 nm); *H NMR (270 MHz, CDC13) 8 9.97 (s, 1H, 10-H), 9.42 (s, 1H, 20-H), 9.19 (d, 1H, J = 4.0 Hz, p~H), 9.03 (d, 1H, J = 4.0 Hz, p-H), 8.98 (d, 1H, J = 4.8 Hz, p-H) 8.89 (d, 1H, J = 4.0 Hz, P-H), 8.70 (d, 1H, J = 4.8 Hz, /?-H), 8.52 (d, 1H, J = 4.8 Hz, P-H), 8.14-8.10 (m, 3H, 5-o/m-Ar), 7.96-7.82 (m, 3H, 5+15-O'm-Ar), 7.50 (s, 1H, NH), 7.34 (m, 2H, 15-m-Ar), 6.48 (m, 1H, 17-H), 6.20 (m, 1H, 18-H), 4.08 (s, 3H, CHs), 2.38 (s, 3H, CH3), -1.89, -2.20 (s, 2H, NH); MALDI-MS m/z 582.6 ([M+Hf, 100%); ES-HRMS calcd. for C35H28N504 ([M+H]+) 582.2141, found 582.2137. Low Rf chlorin regioisomer of Example 11 (7,8-dihydroxy-5-(4-aminophenyl)-15-(4-methoxyphenyl)chlorin) R/= 0.35 (DCM/MeOH, 37:3), mp 182-185°C (decomposed); UV-vis (DCM) Xmm (relative intensity) 410(1.0), 505.5 (0.1), 535 (0.07), 585 (0.04), 636 (0.19) nm; Fluorescence (DCM) A.max 639 nm (X excitation = 410 nm); NMR (270 MHz, 10% DMSO-ds in CDC13) 5 9.96 (s, 1H, 10-H), 9.92 (s, 1H, NH), 9.42 (s, IH, 20-H), 9.22 (m, 1H, P~H), 9.02 (d, 1H, J = 4.8 Hz, p-H), 9.00 (m, 1H, p-H) 8.92 (m, 1H, P~ H), 8.70 (d, 1H, J = 4.8 Hz, P-H), 8.53 (m, 1H, P-H), 8.18-7.91 (m, 6H, 5+15-o/m-Ar), 7.33-7.28 (m, 2H, 15-m-Ar), 6.36 (m, 1H, 7-H), 5.96 (m, 1H, 8-H), 4.10 (s, 3H, CHS), 52 PCT/GBO1/02846 2.30 (s, 3H, CHs), -1.74, -2.17 (s, 2H, NH)\ MALDI-MS m/z 582.6 ([M+H]+, 100%). ES-HRMS calcd. for C35H2gN504 ([M+H]+) 582.2141, found 582.2135.

High Rf-trans bacteriochlorin of Example 12; R/= 0.29 (DCM/MeOH, 37:3:1), mp 152-155°C (decomposed); UV-vis (DCM) A.max (relative intensity) 373.5 (1.0) 514 (0.25), 702 (0.49) nm; Fluorescence (DCM) A,max 708 nm (K excitation = 514 nm); *H NMR (270 MHz, DMSO-d6) 5 10.27 (s, 1H, NH), 9.16 (s, 2H, 10+20-H), 8.96 (d, 2H, J = 4.0 Hz, p-H), 8.24 (d, 2H, J = 4.0 Hz, p-H), 7.99-7.89 (m, 6H, 5+15-o/m-Ar), 7.25 (m, 2H, 15-m-Ar\ 6.30 (m, 2H, 7+17-H), 6.15 (m, 2H, 8+18-H), 5.63 (m, 2H, OH), 5.32 (m, 2H, OH), 3.99 (s, 3H, CH3), 2.20 (s, 3H, CHs), -1-87 (br s, 2H, NH), MALDI-MS m/z 616.3 ([M-H]\ 100%). ES-HRMS calcd. for QjsHsoNsOs ([M+H]+) 616.2196, found 616.2192. Low Rf cw-bacteriochlorin 13; Rf- 0.24 (DCM/MeOH, 19:1), mp 148-151°C (decomposed); UV-vis (DCM) A.max (relative intensity) 373.5 (1.0) 514.5 (0.24), 703 (0.50) nm; Fluorescence (DCM) A.max 708 nm (k excitation =514 nm); 'H NMR (270 MHz, 20% CD30D in CDCI3) 5 9.20 (s, 2H, 10+20-H), 8.78 (m, 2H, P-H), 8.35 (m, 2H, P-H), 8.05 (m, 2H, 5-o-Ar), 7.89-7.86 (m, 3H, 5+15-o/m-Ar), 7.75 (m, 1H, 15-o-Ar), 7.20-7.17 (m, 2H, 15-m-Ar), 6.25 (m, 2H, 7+17-H), 5.86 (m, 2H, 8+18-H), 4.06 (s, 3H, CH3), 2.31 (s, 3H, CH3), (NH's exchanged); MALDI-MS m/z 616.4 ([M+H]+, 100%). ES-HRMS calcd. for C35H30N5O6 ([M+H]+) 616.2196, found 616.2192.

Example 16 5-(4-Isothiocyanatophenyl)-10,15,20-tri(4-methylphosphoniumphenyl)- porphyrin and 5-(4-isothiocyanatophenyI)-15-(4-methyiphosphoniumphenyl)- porphyrin - General Synthetic Procedure Boc N-protected 5-(4-aminophenyl)-10,25,20-tri-(4-carbomethoxyphenyl) porphyrin and 5-(4-aminophenyl)-15-(4-carbomethoxyphenyl) porphyrin were synthesised by mixed condensation using Lindsey conditions (Lindsey, J.S., Schreiman, I.C., Hsu, H.C., Kearney, P.C., Marguerettaz, A.M. (1987) J. Org. Chem. 52, 827) or 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. (1999J Organic Syntheses. 76, 287 - incorporated herein by reference) respectively. The (4-carbomethoxyphenyl) groups on these porphyrins were then converted to (4-(l-bromomethyl)phenyl) groups using the following standard procedure: the porphyrin (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 (MgS04) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted porphyrins, bearing three or one reduced carbomethoxy groups respectively. (4-(l-Hydroxymethyl)phenyl) substituted porphyrins (0.2 mmol) were 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 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 (MgS04). Removal of solvent by evaporation in vacuo afforded the corresponding bromomethyl porphyrins as purple crystalline solids.

Boc N-protected 5-(aminophenyl)-10,25,20-tri-(4-bromomethyiphenyl) porphyrin and 5-(aminophenyl)-15-(4-bromomethylphenyl) porphyrin (0.75 mmol) were dissolved in dry dichloromethane (50 ml) under an atmosphere of argon at 25°C. Triaryl or trialkylphosphine (7.5 mmol) dissolved in dry dichloromethane (10 ml) was 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)-methylphosphonium-meso-aryl porphyrins as lustrous purple crystalline solids. The Boc protecting group was removed by dissolution of the porphyrin 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-(aminophenyl)-methylphosphonium-meso-aryl porphyrins which were converted to the required mono-4-(isothiocyanatophenyl) compounds by treatment 54 - with l,r-thiocarbonyldi-2(lH)-pyridone using standard procedures (Clarke, O.J. and Boyle, R.W. (1999J.C.S. Chem. Commun. 2231).

Example 17 -(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphono-di-ethoxy)phenyl)-porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphono-di-ethoxy)phenyI)- porphyrin - General Synthetic Procedure Boc N-protected 5-(4-aminophenyl)-10,15,20-tri-(4-bromomethylphenyl) porphyrin and 5-(aminophenyl)-15-(4-bromomethylphenyl) porphyrin (0.75 mmol) were dissolved in a mixture of triethyl phosphite (15 mmol) and dry acetonitrile (50 ml). A reflux condenser was fitted and the reaction was refiuxed 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 brine (2 x 20 ml). The organic layer was then dried (MgSCU) 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 purple 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 (Ci8; 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 bromotrimethylsilane (2 equivalents per methylphosphono-di-ethoxy group) for 2 hours followed by reversed phase chromatographic purification chromatography (Cis; gradient elution 0.1 % aqueous TFA to methanol) (McKenna, C.E., Higa, M.T., Cheung, N.H., McKenna, M-C. (1977) 2, 155). Boc deprotection (see above) followed by conversion of the unmasked 4-(aminophenyl) group to its isothiocyanato analogue was performed using standard procedures (Clarke, O.J. and Boyle, R.W. (1999 J.C.S. Chem. Commun. 2231). 55 Example 18 -(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphonato-di-ethoxy)phenyI)- 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) porphyrin and 5-(aminophenyI)-15-(4-hydroxymethylphenyl) porphyrin (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) porphyrins. Treatment with aqueous sodium hydroxide (1M) gave the sodium salts of tri or mono ((4- methylphosphonatoethoxy)phenyl) porphyrins (Boyle, R.W. and vanLier, J.E. (1995) Synthesis 1079). Boc deprotection and generation of the isothiocyanato group were performed as described above.

Example 19 5-(4-Isothiocyanatophenyl)-10,15,20-tri((4- methylpyridiniumyl)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylpyridiniumyl)phenyl)- porphyrin - General Synthetic Procedure Boc N-protected 5-(aminophenyl)-10,25,20-tri-(4-bromomethylphenyl) porphyrin and 5-(aminoophenyi)-15-(4-bromomethylphenyl) porphyrin (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 (C18; gradient elution 0.1% aqueous TFA to methanol) to yield the N-Boc protected 4-arninophenyl compounds. Deprotection of the aminophenyl group(s) and conversion to the isothiocyanato analogue(s) were conducted using the standard protocols (see above). 56 Example 20 5-(4-Isothiocyanatophenyl)-15-3aryl-10,20-(l,2-dihydroxyethyl)-porphyrin - General Synthetic Procedure The Fmoc protected 5-(4-aminophenyl)-15-aryl porphyrin (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-diarylporphyrin as a purple 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-diarylporphyrin (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 porphyrin as a purple crystalline solid. Zinc 5-(Fmoc aminophenyl)-15-aryl- 10,20-diethenyl porphyrin 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 porphyrin 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. Porphyrins 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 porphyrin, 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) porphyrin, 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- 57 bis(l,2-dihydroxyethyl) 7,8-dihydroxychlorin and 5-(Fmoc aminophenyl)-15-aryl-10,20-bis(l,2-dihydroxyethyl) 7,8,17,18-tetrahydroxybacteriochiorin 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.JC.S. Chem. Commun. 2231).

Example 21 5-(4-IsothiocyanatophenyI)-15-phenyl-10,20-(diaryl)-porphyrins -Synthesis from 5,15-diphenyI porphyrins by Pd° mediated Suzuki coupling Boc N-protected 5-(aminophenyl)-15-phenyI porphyrin was brominated at the 10 and 20 meso positions as described above. The meso-10,20-dibrominated product (0.75 mmol) was dissolved in dry THF (50 ml) or toluene (50 ml), depending upon the boronic acid used in the coupling reaction, tetrakis-(triphenylphosphine) palladium (0) (0.75 mmol) and anhydrous potassium phosphate (0.75 mmol) were added, a reflux condenser was then fitted to the flask and the whole apparatus was placed under an atmosphere of argon. The required aryl or heterocyclic boronic acid was then added as a solution in the appropriate solvent (10 ml) by injection. The reaction was brought to reflux and followed to completion by TLC. On completion the crude reaction mixture was diluted with dichloromethane (100 ml) and extracted with saturated sodium bicarbonate (2 x 50 ml), water (2 x 50 ml) and brine (2 x 50 ml). The organic phase was dried (Mg SO4) and concentrated by evaporation in vacuo. Finally, the residue was purified by flash column chromatography (silica; gradient elution: dichloromethane to methanol) to give the Boc N-protected 5-(aminophenyl)-15-phenyl-10,20-(diaryl)-porphyrin as a purple crystalline solid. The Boc protecting group was removed by dissolution of the porphyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (1.2 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-(aminophenyl)-15-phenyl-10,20-(diaryl)-porphyrin which was converted to the title compound by treatment with 1, r-thiocarbonyldi-2(lH)-pyridone using standard procedures (Clarke, O.J. and Boyle, R.W. (1999 JC.S. Chem. Commun. 2231).

Example 22 5-(4-Isothiocyanatophenyl)-10,15,20-tri(4-glycosylphenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-l5-(4-glycosylphenyI)- porphyrin - General Synthetic Procedure 4-(2\3 \4',6'-tetra-0-acetyl-p-D-glucopyranosyloxy)benzaldehyde was condensed with 4-nitrobenzaldehyde and pyrrole using Lindsey conditions (Sol, V., Blais, J.C., Carre, V., Granet, R„ Guilloton, M., Spiro, M., Krausz, P. (1999) J. Org. Chem. 64, 4431) and the crude reaction mixture purified by flash column chromatography to give 5-(4-nitrophenyl)-10,15,20-tris[4-(2',3',4,,6'-tetra-0-acetyl-(3-glucopyranosyloxy)phenyl] porphyrin. Alternatively, 4-(2',3\4\6'-tetra-0-acetyl-(3-D- glucopyranosyioxy)benzaldehyde was used to synthesise 5-(4-(2',3',4\6'-tetra-0-acetyl-(3-D-glucopyranosyloxy)phenyI) dipyrromethane using the method of Boyle (Boyle, R.W., Bruckner, C., Posakony, J., James, B.R., Dolphin, D. (1999J Organic Syntheses. 76, 287) which was then condensed to give 5-(4-nitrophenyl)-15,-[4-(2',3',4',6'-tetra-0-acetyl-P-glucopyranosyloxy)phenyl] porphyrin. Reduction of the nitro group of these porphyrins 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 porphyrins, 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 (MgSCU). Purification by flash column chromatography gave the Fmoc N-protected 5-(4-aminophenyl)-l 0,15,20-tris[4-(2' ,3' ,4' ,6' -tetra-0-acetyl-(3-glucopyranosyloxy)phenyl] porphyrin or 5-(4-aminophenyl)-15,-[4-(2',3',4',6'-tetra-0-acetyl-p-glucopyranosyloxy)phenyl] porphyrin. N and O protecting groups were removed by dissolution of the porphyrin in dichloromethane/morpholine (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 porphyrin was recovered by precipitation with hexane. Finally, the 59 - -(4-aminophenyl) porphyrin was dissolved in dry methanol and lj'-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 (Ci&;gradient elution: 0.1% aqueous TFA to methanol).

Example 23 : Symmetrical Porphyrin/ Chlorin Diol/ Bacteriochlorin Tetrol Series ,15-(3,4,5- Trismethoxyphenyl)porphyrin (A general procedure) To a 3 L round bottom flask was added 5-(3,4,5-trismethoxyphenyl)dipyrromethane (1.86 g, 6 mmol), then DCM (1L) under N2. To this stirred solution was added trimethylorthoforraate (48 ml, mmol). A pressure equalizing dropping fUnnel containing a solution of trichloroacetic acid (23.0 g, mmol) in DCM (500 ml) was then fitted to the flask and the solution added dropwise to the reaction mixture over a period of 10 min. The reaction vessel was covered in aluminium foil to exclude light and allowed to stir under N2 for a period of 3.5 h. Pyridine was then added to the reaction mixture, rapidly with stirring, and the reaction allowed to stir for a further 16 h. at room temperature under N? with the light excluded. The aluminium foil was removed and air was bubbled through the solution for a period of 20 min. After this the reaction was left to stir unstoppered for a further period of 3 h. at room temperature with the aluminium foil removed. The reaction mixture was then concentrated in vacuo to remove DCM and remaining pyridine by evaporator, then high vacuum. The crude reaction mixture was then chromatographed on flash silica-gel (250 ml), (dry loaded on to 50 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting with chloroform . The title compound was obtained as purple crystalline solid (347 mg, 18%); XmJ (relative intensity) 410 (1.0), 502 (0.04), 538(0.02), 578 (0.015), 630 (0.01) nm; UV-VIS (CH2CI2) (fluorescence) XmAX = 634 nm (A, excitation = 408 nm); (270 MHz, CDCI3) 10.32 (2H, s, 10-H, 20-H), 9.40 (4H, d, J = 4.8 Hz, /3-H), 9.18 (4H, d, J = 4.8 Hz, P-H), 7.52 (4H, s, o-Ar), 4.20 (6H, s, CHs), 4.00 (12H, s, CH3), -3.10 (2H, br. s, NH); MS (MALDI) m/z = 643.4 (100%, M+). 60 - 7,8-Dihydroxy 5,15-(3,4,5-trismethoxyphenyl) chlorin (A general procedure) To a stirred solution of 5,15-(3,4,5-trismethoxyphenyl)porphyrin (50 rag, 77.8 |imol) in HPLC grade chloroform (5.0 ml) was added a solution, in anhydrous pyridine (0.5 ml), of osmium tetroxide (2.5 eq0.195 mmol, 49 mg). The reaction vessel was flushed with N2 and sealed with a lightly greased glass stopper, then covered in aluminium foil to exclude the light and left to stir for 72 h at room temperature. After this period the reaction vessels glass stopper was replaced with a plastic stopper and a continuous stream of hydrogen sulfide gas was bubbled through the reaction mixture for 5 min., (a gas outlet needle was attached and allowed excess hydrogen sulfide gas to escape into a series of Dreshel bottles filled with mineral oil and a bleach solution respectively). After this time the reaction mixture was filtered through Celite® and then concentrated in vacuo. Any excess pyridine was removed under high vacuum. The crude reaction mixture was then chromatographed on flash silica-gel (100 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting with 1% methanol in DCM. Some starting material was recovered (15%) and the title compound was obtained as browny-purple crystalline solid , (26 mg, 50%); m.p. 170 °C (decomposed); UV-VIS (CH2CI2) Xmax (relative intensity) 410 (1.0) 504 (0.09), 534 (0.06), 582 (0.04), 636 (0.18) nm; UV-VIS (CH2CI2) (fluorescence) A.max 639 nm (X excitation 410 nm); 5H(270 MHz, CDC13) 9.98 (1H, s, 10-H), 9.42 (1H, s, 20-H), 9.20 (1H, m, /3-H), 9.04 (1H, d, J = 4.0 Hz, (3-H), 8.99 (2H, s, /3-H), 8.79 (1H, d, J = 4.0 Hz, /3-H), 8.66 (1H, m, /3-H), 7.45 (1H, d, J = 1.6 Hz, 15-o-Ar), 7.42 (1H, d, J = 1.6 Hz, 15-o-Ar), 7.40 (1H, d, J = 1.6 Hz, 5-o-Ar), 7.19 (1H, d, J= 1.6 Hz, 15-o-Ar), 6.49 (1H, d, J= 7.3 Hz, 7-H), 6.23 (1H, d, J = 7.3 Hz, 8-H), 4.17 (3H, s, CH3), 4.15 (3H, s, CHS), 4.04 (3H, s, CH3), 4.00 (3H, s, CH3\ 3.98 (3H, s, CH3\ 3.91(3H, CH3\ -1.80 (1H, br. s, NH), -2.19 (1H, br. s, NH), (OH's not observed); MS (MALDI) m/z = 677.3 (100%, M+); HRMS calcd. for C38H,6N40s: 676.2533. Found: 676.2587. 7,8,17,18-Tetrahydroxy5,15-(3,4,5-trismethoxyphenyl) bacteriochlorin (A general procedure) To a stirred solution of 5,15-(3,4,5-trismethoxyphenyl)porphyrin (50 mg, 77.8 jamol) in HPLC grade chloroform (5.0 ml) was added a solution, in anhydrous pyridine (0.5 ml), of 61 osmium tetroxide (5.0 eq., 0.39 mmol, 49 mg). The reaction vessel was flushed with N2 and sealed with a lightly greased glass stopper, then covered in aluminium foil to exclude the light and left to stir for 72 h at room temperature. After this period the reaction vessels glass stopper was replaced with a plastic stopper and a continuous stream of hydrogen sulfide gas was bubbled through the reaction mixture for 5 min., (a gas outlet needle was attached and allowed excess hydrogen sulfide gas to escape into a series of Dreshel bottles filled with mineral oil and a bleach solution respectively). After this time the reaction mixture was filtered through Celite® and then concentrated in vacuo. Any excess pyridine was removed under high vacuum. The crude reaction mixture was then chromatographed on flash silica-gel (100 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting initially with 1% methanol in DCM to elute chlorin by-product then 2.5 % methanol in DCM to elute the major bacteriochlorin isomer (assumed as trans form Bruckner et al (1995) Tetrahedron Lett. 36, 9425). The title compound was obtained as a greeny-pink crystalline solid, (20 mg, 36%); m.p. 135°C (decomposed); UV-VIS (CH2C12) A.max (relative intensity) 374 (1.0) 512 (0.23), 702 (0.52) nm; UV-VIS (CH2C12) (fluorescence) Xmax 708 nm (X excitation 512 nm); SH(270 MHz, CDCI3) 9.23 (2H, s, 10-H, 20-H), 8.79 (2H, d, J = 3.2 Hz, (3-H), 8.44 (2H, d, J = 3.2 Hz, /3-H), 7.37 (2H, s, 5+15-o-^r), 7.13 (2H s, 5 + 15-o-Ar\ 6.31 (2H, d, J = 6.5 Hz, 7-H, 17-H), 6.01 (2H, d, J = 6.5 Hz, 8-H, 18-H), 4.12 (6H s, CHS), 3.92 (6H, s, CH3), 3.89 (6H, s, CH3), -1.97 (2H, br. s, NH), (OH's not observed); MS (MALDI) m/z = 712.4 (100%, (M+l)+); HRMS calcd. for C38H36N4O10: 710.2590. Found: 710.2607.

Example 24 : Unsymmetrical Porphyrin/ Chlorin Diol/ Bacteriochlorin Tetrol Fluorochrome Sets for Bioconjugation 5-(4-Acetomiclophenyl)-15-(4-methoxyphenyl)porphyrin The required unsymmetrical diphenylporphyrin was synthesised using the general procedure outlined earlier, but with only slight modification. In this example a mixture of dipyrromethanes were used. Due to the different reactivities of the respective dpyrromethanes, the amounts needed for optimisation of mixed porphyrin were different. For the same scale reaction 5-(4-methoxyphenyl)dipyrromethane (505 mg, 2 mmol) and 62 -(4-acetomidophenyl)dipyrromethane (838 mg, 3 mmol) were used. The porphyrin mixture was chromatographed on silica-gel (400 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting initially with DCM (1 glass pipette full of triethylamine was added to 500 ml of eluent to aid elution) to remove 5,15-(methoxyphenyl)porphyrin byproduct. After separation of this component the elution was continued with chloroform to allow 5-(4-Acetomidophenyl)-15-(4-methoxyphenyl)porphyrin collection. The desired porphyrin was obtained as purple crystals; (150 mg, 12%); lmax/ (relative intensity) 410 (1.0), 502 (0.04), 538 (0.02), 578 (0.015), 630 (0.01) nm; 8H(270 MHz, CDC13) 10.35 (2H s, 10-H, 20-H), 9.43 (4H, d, J = 4.8 Hz, 0-H), 9.14 (4H, d, J = 4.8 Hz, p-H), 8.65 (2H, d, J = 7.2 Hz, 5-m-Ar), 8.22-8.12 (4H d (overlapping), J = 8.1 Hz, 5-o-Ar+15-o-Ar), 7.56 (2H, d, J = 8.1 Hz, 15-m-Ar), 4.14 (3H, s, CHj), -3.00 (2H, br. s, NH); MS (MALDI) m/z = 550.3 (100%, M+). -(4-Aminophenyl)-15-(4-methoxyphenyl)porphyrin -(4-Acetomidophenyl)-15-(4-methoxyphenyl)porphyrin (100 mg, 0.182 mmol) was treated with 18% hydrochloric acid (200 ml) and fitted with an air condenser. The green solution was left to warm to 85°C for a period of 3 h. Prior to cooling, the reaction mixture was concentrated in vacuo (water aspirator; evaporator water bath at 75°C) to remove excess hydrochloric acid then treated carefully with a solution of triethylamine (50 ml) in DCM. The organic extract was washed with water (100 ml) then saturated brine (100 ml) prior to drying (ctnhyd. NaiSCU), filtering via Buckner funnel and finally concentration in vacuo. The required porphyrin was obtained by chromatography on silica-gel (100 ml), (liquid loaded in 10 ml DCM) eluting with DCM (1 glass pipette full of triethylamine was added to 500 ml of eluent to aid elution). The desired porphyrin was obtained as purple crystals; (150 mg, 12%); km.J (relative intensity) 410 (1.0), 503 (0.045), 538(0.02), 578 (0.015), 630 (0.005) nm; UV-VIS (CH2C12) (fluorescence) ^max = 634 nm (X excitation = 410 nm); SH(270 MHz, CDCI3) 10.30 (2H, s, 10-H, 20-H), 9.39 (4H, d, J = 4.9 Hz, p-H), 9.17 (2H, d, J = 4.9 Hz, p-H), 9.10 (2H, d, J = 4.9 Hz, /?-#), 8.19 (2H, d, J = 8.8 Hz, 15-o-Ar), 8.07 (2H, d, J = 8.1 Hz, 5-o-Ar), 7.35 (2H, d, J = 8.8 Hz, 15-m-Ar), 7.14 (2H, d, J = 8.1 Hz, 5-m-Ar), 4.13 (3H, s, CH3), 4.08 (2H, br. s, NH), -3.06 (2H br. s, NH); MS (MALDI) m/z = 508.3 (100%, (M+l)*). 63 -(4-Fluoranomethylaminophenyl)-15-(4-m£thoxyphenyl)porphyrin To a stirred solution of 5-(4-aminophenyI)-15-(4-methoxyphenyl)porphyrin (28 mg, 55 prnol) in anhydrous 1,4-dioxane (2.5 ml) was added solid sodium hydrogen carbonate (6 eq., 2S mg, 0.33 mmol). To this mixture was then added a solution of 9-fluorenomethylchloroformate (2 eq., 0.11 mmol, 28.5 mg) in 1,4-dioxane (0.5 ml) under N?. 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 had gone to completion (as monitored by TLC). The 1,4-dioxane was removed in-vcicuo 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 (atihyd. Na2SC>4), filtered and concentrated in vacuo. The required porphyrin was obtained by chromatography on silica-gel (100 ml), (dry loaded on to 10 ml flash silica-gel from DCM and a little methanol for solubility) eluting with DCM. The desired porphyrin was obtained as purple crystals; (38 mg, 95%); XmaJ (relative intensity) 410 (1.0), 505 (0.042), 541 (0.02), 578 (0.015), 633 (0.01) nm; UV-VIS (CH2CI2) (fluorescence) A,max = 635 nm (A, excitation = 410 nm); 5H(270 MHz, CDCI3) 10.35 (2H, s, 10-H, 20-H), 9.69 (1H, br. s,M), 9.44 (4H, d, J = 4.8 Hz, fi-H), 9.12 (4H, d, J = 4.8 Hz, JB-H), 8.20-8.17 (4H 2 x d (overlapping), J = 8.1 Hz, 5+15-o-Ar), 7.85 (4H 5+15-m-Ar), 7.76-7.66 (2H m, fluoreno-Ar), 7.51-7.30 (6H m,Jlureno-Ar), 4.69 (2H, d, J = 7.2 Hz, CH2), 4.30 (1H t, J = 7.2 Hz, CH), 4.13 (3H s, CH3), -3.15 (2H, br. s, NH); MS (MALDI) m/z = 731.5 (100%, (M+l)+), 508.3 (52%, (M-FMOC+lf). 17,18-Dihydroxy-5-(4-aminophenyl)-l 5-(4-methoxyphenyl) chlorin and 7,8-dihydroxy 5-(4-aminophenyl)-15-(4-methoxyphenyl) chlorin regioisomers 5-(4-aminophenyl)-15-(4-methoxyphenyl)porphyrin (28 mg, 55.2 |amol) was converted to a mixture of chlorin diol regioisomers using the general chlorin formation procedure given earlier. The crude reaction mixture was then chromatographed on flash silica-gel (200 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting with 1% methanol in DCM to elute first some unreacted starting material then the higher Rf chlorin isomer as a browny-purple 64 crystalline solid. The lower Rf isomer was obtained by further elution with 2.5% methanol in DCM and gave also a browny-purple crystalline solid.

High Rf chlorin regioisomer (2,3-dihydroxy-5-(4-methoxyphenyl)-15-(4-aminophenyl) chlorin, from nOe measurements and JPP paper): (8.5 mg, 30%); m.p. 165°C (decomposed); UV-VIS (CH2CI2) Amax (relative intensity) 401 (0.99), 414 (1.0), 503 (0.08), 535 (0.07), 582 (0.035), 636 (0.22) nm; UV-VIS (CH2CI2) (fluorescence) W 639 nm (K excitation 412 nm); 5H(270 MHz, 10% MeOH-d4 in CDCI3) 9.95 (1H, s, 10-H), 9.42 (IH, s, 20-H), 9.17 (1H, d, J =4.8 Hz, P-H), 9.03 (1H, d, J=4.0 Hz, (3-H), 8.97 (2H, s, p-H) 8.78 (IH, d, J = 4.8 Hz, p-H), 8.51 (IH, d, J = 4.8 Hz, p-H), 8.05 (2H, d, J = 8.9 Hz, o-Ar), 7.94 (2H, d, J = 8.1 Hz, o -Ar), 7.25 (2H, d, J = 8.9 Hz, m-Ar), 7.12 (2H, d, J = 8.1 Hz, m '-Ar), 6.42 (IH, d, J = 6.5 Hz, 17-H), 6.03 (IH, d, J = 6.5 Hz, 18-H), 4.08 (3H, s, CHs), (NH's exchanged), (OH's not observed).; MS (MALDI) m/z = 642.2 (100%, (M+l)+).

Low Rf chlorin regioisomer (2,3-dihydroxy-5-(4-aminophenyl)-l5-(4-methoxyphenyl) chlorin from nOe measurements and JPP paper): (8.5 mg, 30%); m.p. 168°C (decomposed); UV-VIS (CH2C12) W (relative intensity) 401(0.99), 413 (1.0), 507 (0.08), 536 (0.06), 586 (0.025), 637 (0.20) nm; UV-VIS (CH2C12) (fluorescence) W 639 nm (K excitation 412 nm); 5H(270 MHz, 10% MeOH-cLt in CDCI3) 9.96 (IH, s, 20-H), 9.40 (IH, s, 10-H), 9.18 (1H, d, J =4.8 Hz, P-H), 9.05 (IH, d, J=4.8 Hz, P-H), 8.98 (1H, d, J = 4.0, p-H) 8.92 (IH, d, J = 4.0 Hz, P-H), 8.74 (IH, d, J = 4.0 Hz, P-H), 8.58 (IH, d, J = 4.0 Hz, p-H), 8.13 (IH, d, J = 8.9 Hz, o-Ar), 8.08 (IH, d, J = 8.9 Hz, o-Ar), 7.95 (IH, d, J = 8.1 Hz, o'-Ar), 7.79 (IH, d, J = 8.1 Hz, o'-Ar), 7.36 (IH, d, J = 8.9 Hz, m-Ar), 7.30 (IH, d, J = 8.9 Hz, m-Ar), 7.11 (IH, d, J = 8.1 Hz, m '-Ar), 7.05 (IH, d, J = 8.1 Hz, m '-Ar), 6.42 (IH, d, J = 6.5 Hz, 7-H), 6.09 (IH, d, J = 6.5 Hz, 8-H), 4.11 (3H, s, CHS), (NH's exchanged), (OH's not observed); MS (MALDI) m/z = 642.2 (100%, (M+i n 65 17,18-Dihydroxy-5-(4-isothiocyanatophenyl)-15-(4-methoxyphenyl)chlorin (higher Rf regioisomer) To a stirred solution of l,r-thiocarbonyldi-2(l //)-pyridone (1.07 eq., 7.6 mg, 43.5 mmol) in DCM (25 ml), (dried via passage through an activated alumina column) was added a solution of 17,18-dihydroxy-5-(4-methoxyphenyl)-15-(4-aminophenyl)chlorin (17.5 mg, 23.2 Dmol) in DCM (10 ml). The reaction flask was covered with aluminium foil to exclude light and left to stir under N2 for 2 h, at which time TLC indicated complete loss of starting material. The reaction mixture was then washed with water (2 x 50 ml) and saturated brine (50 ml) then dried (cinhyd. NazSCU). The organic extract was then filtered and concentrated on to 10 ml flash silica-gel and chromatographed on flash silica-gel (100 ml), eluting with 1% methanol in DCM to elute the required isothiocyanato chlorin diol (NB. traces of all TDP must be removed prior to concentration of product from column chromatography otherwise some decomposition to 3 higher Rf byproducts occurs. These have not been identified at this time). The title compound was isolated as a browny-purple crystalline solid (17 mg, 90%); m.p. 155°C (decomposed); UV-VIS (CH2CI2) A.max (relative intensity) 410 (1.0) 505 (0.09), 534 (0.06), 586 (0.04), 637 (0.18) nm; UV-VIS (CH2CI2) (fluorescence) Xmay 639 nm (X excitation 412 nm); SH(270 MHz, 10% MeOH-cLt in CDCI3) 10.0 (IH, s, 10-H), 9.45 (IH, s, 20-H), 9.20 (IH, d, J =4.8 Hz, /3-H), 9.06 (IH, d, J=4.0 Hz, /3-H), 9.02 (1H, d, J = 4.8 Hz, /3-H) 8.84 (IH, d, J = 4.8 Hz, /3-H), 8.64 (IH, d, J = 4.0 Hz, /3-H), 8.55 (IH, d, J = 4.8 Hz, /3-H), 8.21 (IH, d, J = 8.1 Hz, o-Ar), 8.15 (IH, d, J = 8.1 Hz, o-Ar), 8.05 (IH, d, J = 8.9Hz, o'-Ar) 7.93 (IH, d, J = 8.9 Hz, o '-Ar), 7.65 (2H, m, m-Ar), 7.24 (2H, m, m -Ar), 6.43 (IH, d, J = 6.5 Hz, 17-H), 6.04 (IH, d, J = 6.5 Hz, 18-H), 4.08 (3H, s, CHS), (NH's exchanged), (OH's not observed); MS (MALDI) m/z = 583.7 (100%, M+); HRMS calcd. for C34H26N5O3S: 584.1757. Found: 584.1756 ((M+l)+). 7,8,17,18-Tetrahydroxy-5-(4-fluorenomethylaminophenyl)-l 5-(4-methoxyphenyl) bacteriochlorin (cis/trans stereoisomers) -(4-Fluorenomethylaminophenyl)-15-(4-methoxyphenyl)porphyrin (35 mg, 48.0 Dmol) was converted to a mixture of bacteriochlorin stereoisomers using the general bacteriochlorin formation procedure given earlier. The crude reaction mixture was then 66 PCT/GBO1/02846 chromatographed on flash silica-gel (200 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little methanol to ensure complete solubility) eluting initially with 1% methanol in DCM to elute the higher Rf chlorin byproducts, then 2% methanol/ DCM to elute separately the two stereoisomeric bacteriochlorins. The higher Rfirans bacteriochlorin isomer was isolated as a pinky-green crystalline solid, (6 mg, 15%); m.p. 14>C. (decomposed); UV-VIS (CH2CI2) Xmax (relative intensity) 374 (1.0) 512 (0.23), 702 (0.52) nm; UV-VTS (CH2CI2) (fluorescence) X.max 708 nm (k excitation 512 nm); 5H(270 MHz, 10 % MeOH-d4 in CDCI3) 9.20 (2H, s, 10-H, 20-H), 8.78 (2H, d, J =4.0 Hz, (3-#X 8.36 (2H, d, J = 4.0 Hz, p-H), 7.95 (2H, m, o-Ar), 7.85 (2H, d, J = 7.3 Hz, fhioreno-Ar), 7.79 (2H, m, o'-Ar), 7.65 (2H, m, mAr), 7.47-7.38 (6H, m, fluoreno-Ar), 7.24 (2H, m, m-Ar), 6.27-6.24 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H 17-H), 5.85 (2H, d, J = 6.5 Hz, 8-H, 18-H), 4.65 (2H, d, J = 7.2 Hz, CH2\ 4.39 (1H, t, J = 7.2 Hz, CH), 4.06 (3H, s, CH;), -1.94 (2H, br. s (partly exchanged), NH), (OH's not observed); MS (MALDI) m/z = 800.4 (100%, (M+lf).

The lower Rt- crs-bacteriochlorin isomer was isolated as a pinky-green crystalline solid, (8.5 mg, 21%); m.p. 148°C (decomposed); UV-VTS (CH2CI2) A^ax (relative intensity) 374 (1.0) 512 (0.24), 703 (0.54) nm; UV-VTS (CH2C12) (fluorescence) Amax 708 nm (X excitation 512 nm); 5H(270 MHz, 10 % MeOH-d4 in CDCI3) 9.12 (2H, s, 10-H, 20-H), 8.76 (2H, d, J =4.8 Hz, P-i7), 8.34 (2H, 2 x d (overlapping), J = 4.8 Hz, P-H), 8.02 (2H, m, o-Ar), 7.85 (2H, d (obscurred), J = 8.0 Hz, 0 -Ar), 7.83 (2H, d, J = 7.3 Hz,.fluoreno-Ar), 7.76 (2H, d, J = 8.0 Hz, m '-Ar), 7.50-7,38 (6H, m,fluoreno-Ar), 7.24 (2H, m, m-Ar), 6.27-6.23 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H, 17-H), 5.85-5,82 (2H, 2 x d (overlapping), J = 6.5 Hz, 8-H, 18-H), 4.65 (2H, d, J = 7.2 Hz, CH2), 4.39 (IH, t, J = 7.2 Hz, CH), 4.05 (3H, s, CH3), -1.88 (2H, br. s (partly exchanged), NH), (OH's not observed); MS (MALDI) m/z = 800.4 (100%, (M+l)+). 67 7,8.17,18-Tetrahydroxy-5-(4-isothiocyanatophenyl)-15-(4-methoxyphenyl) bacteriochlorin (lower Rf cis stereoisomer) A solution of 7,8,17,18-tetrahydroxy-5-(4-fluorenomethyaminophenyl)-l 5-(4-methoxyphenyl) bacteriochlorin (lower Rf cis stereoisomer), (8.5 mg, 10.7 pimol) in 25% methanol in DCM (1.25 ml) was treated with piperidine (50 eq., 53 (^1, 0.53 mmol) and left to stir for a period of 3 h. at room temperature under N2 with the light excluded. The reaction mixture was concentrated in vacuo to remove all traces of piperidine (high vacuum needed). To a stirred solution of l,r-thiocarbonyldi-2(l #)-pyridone (1.07 eq., 7.6 mg, 43.5 mmol) in DCM (25 ml), (dried via passage through an activated alumina column) was added a solution of 2,3,12,13-tetrahydroxy-5-(4-aminophenyl)-15-(4-methoxyphenyl)bacteriochlorin (6.1 mg, 10,7 ^mol) in DCM (10 ml). The reaction flask was covered with aluminium foil to exclude light and left to stir under N2 for 2 h, at which time TLC indicated complete loss of starting material. The reaction mixture was then washed with water (2 x 50 ml) and saturated brine (50 ml) then dried (anhyd. Na^O-i). The organic extract was thenjfiltered and concentrated on to 10 ml flash silica-gel and chromatographed on flash silica-igel (100 ml), eluting with 2% methanol in DCM to elute the required isothionato bacteriochlorin tetrol (NB. traces of all TDP must be removed prior to concentration of product from column chromatography otherwise some decomposition occurs). The lower Rf czs-bacteriochlorin isomer was isolated as a pinky-green crystalline solid, (5.0 mg, 76%); m.p. 132°C (decomposed); UV-VIS (CH2CI2) Amax (relative intensity) 375 (1.0) 516 (0.22), 702 (0.48) nm; UV-VIS (CH2CI2) (fluorescence) Wc 709 nm (X excitation 516 nm); 5H(270 MHz, 10 % MeOH-d4 in CDCI3) 9.20 (IH, s, meso-H), 9.18 (IH, s, meso -H), 8.77 (2H, d, J =4.8 Hz, /3-H), 8.40 (IH, d, J = 4.8 Hz, P-H), 8.34 (IH, d, J = 4.8 Hz, P-H), 8.14 (2H, m, o-Ar), 8.05 (2H, m, o'-Ar), 7.42-7.08 (4H, m, 5+ 15-m-Ar), 6.20 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H, 17-H), 5.98 (1H, d, J = 6.5 Hz, 8-H), 5.93 (IH, d, J= 6.5 Hz, 18-H), 4.04 (3H, s, CH,), -1.80 (2H, br. s (partly exchanged), NH), (OH's not observed); MS (MALDI) m/z = 618.9 (100%, (M+l)+); HRMS calcd. for Cj-dtwNsOjS: 618.1815. Found: 618.1810 ((M+l)+).

Further synthetic protocols and methodology protocols are also described in Sutton et al, Porphyrin, Chlorin and Bacteriochlorin Isothiocvanates - Synthesis and 68 PCT/GBO1/02846 Potential Applications in Fluorescence Imaging and Photodynamic Therapy (Journal of Phthalocyanines & Photosensitisers - in press) and in Oliver J Clarke, Isothiocyanato Porphyrins for bioconjugation : synthesis and applications in photochemotherapv and fluorescence imaging (PhD thesis, April 2001, University of Essex); the entire contents of each of which are incorporated herein by reference.

Methodology Description 1 : General Bioconiligation protocol Hexahydroxy PITC + Antibody A stock solution of hexahydroxy PITC 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 |iL aliquots.

A 1 M solution of sodium bicarbonate was prepared and adjusted to pH 9.0 with 2 M sodium hydroxide.

To a 250 |iL aliquot of antibody was added 30 (iL of 1 M sodium bicarbonate. A predetermined volume of hexahydroxy PITC stock solution was then added to give a desired molar ratio (MR) of porphyrin to antibody. For example an MR of 20 was achieved via the addition of 10 jiL of stock solution to 250 jj.L 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 |iL 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 250 |jL 10 mg/mL 30 pL 10 |xL 15 (jL 250 |llL 10 mg/mL 30 |xL 5 (oL 20 |iL 250 nL 10 mg/mL 30 |aL 2.5(jL 22.5 \xL 69 2.5 250 jlL 10 mg/mL 30 ^L 1.25 |iL 23.75 |iL 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-equiiibrated with sterile PBS (25 mL). The column was eiuted with sterile PBS. Antibody-porphyrin conjugate was eiuted in the first coloured band/fraction. The antibody-porphyrin conjugate concentration following dilution during chromatography was determined as 1.25 mg/mL. The degree of labelling (DOL) of porphyrin to antibody was calculated via standard spectroscopic methods using known constants of molar absorptivity for both porphyrin and protein.

Antibody-porphyrin conjugates were stored, without further concentration, in PBS + azide at 0°C unless otherwise stated. iV-Methylpyridinium chloride PITC + Antibody A stock solution of TV-methylpyridinium chloride PITC in DMSO was prepared to a molarity of 0.027, this solution was desiccated and stored at 09C 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 |iL aliquots.

To a 250 p.L aliquot of antibody was added 250 pL of sterile PBS then 60 jiL of 1 M sodium bicarbonate. A predetermined volume of /V-methylpyridinium chloride PITC stock solution was then added to give a desired molar ratio (MR) of porphyrin to antibody. For example an MR of 20 was achieved via the addition of 10 |j.L of stock solution to 500 |_iL of antibody at 5 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 uL with further portions of DMSO, 70 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 500 pL mg/mL 60 pL pL pL 500 pL mg/mL 60 pL pL pL . 500 pL mg/mL 60 pL 2.5pL 22.5 pL 2.5 500 pL mg/mL 60 pL 1.25 pL 23.75 pL Table 2.0 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 eiuted with sterile PBS. Antibody-porphyrin conjugate was eiuted in the first coloured band/fraction. The antibody-porphyrin conjugate concentration following dilution during chromatography was determined as 1.25 mg/mL. The degree of labelling (DOL) of porphyrin to antibody was calculated via standard spectroscopic methods using known constants of molar absorptivity for both porphyrin and protein.

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

Methodology Description 2 : Standard Photocvtotoxicitv Cells are grown to confluence or appropriate density then washed 2 times with PBS (phosphate buffered saline) to eliminate all trace of FBS (foetal 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 71 PCT/GBO1/02846 medium (without FBS )to eliminate unbound photosensitiser, then resuspended and seeded in 96 wells plates (1x10" cells/well) in quadruplate.Plates are then either irradiated (3.6J/cm2 of filtered red light D600nm ) 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 jj.1 of MTT solution (Sigma Thiazolyl blue, 4.8xl0"4M 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 (lOOjil/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 Cytometry Chromophore Analysis The two fluorochromic probes were generated from separate reactions of 2,3-dihydroxy-5-(4-methoxyphenyl)-15-(4-isothiocyanatophenyl)chlorin (higher Rf regioisomer) and 2,3,12,13-tetrahydroxy-5-(4-isothionatophenyl)-15-(4-methoxyphenyl) bacteriochlorin (lower Rf 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 72 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-methoxyphenyl)-15-(4-acetomidophenyl)chiorin (higher Rf regioisomer) and 2,3,12,13-tetrahydroxy-5-(4-acetomidophenyl)-15-(4-methoxyphenyl) bacteriochlorin (higher Rf 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-methoxyphenyl)-15-(4-acetomidophenyl)chlorin (higher Rf regioisomer) than with 2,3,12,13-tetrahydroxy-5-(4-acetomidophenyl)-15-(4-methoxyphenyl) bacteriochlorin (higher Rf 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 incorporation into the HeLa cell membrane resulting in restoration of 73 PCT/GBO1/02846 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-Electroe.liiter • 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). 74 • 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 lO21 in FACS tube (Falcon 2054) and wash with lmL PBS by centrifugation (400g 3min) and resuspension by agitation Block cells in 500 jiL 2% Marvel milk powder in PBS, 1% BSA 30 min R.T Wash cells in 1 mL PBS/BSA/Azide centifuge and resuspend pellet (as above) Add 10 |iL appropriate antibody dilution. Incubate on ice Ih Wash cells in 1 mL PBS/BSA/Azide centifuge and resuspend pellet (as above) Add 50 jiL 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 \iL PBS/BSA/Azide.

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

PBS/BSA/AZIDE 250 mL PBS 75 0.625g BSA 1.56 mL Sodium Azide (1.6M) Methodology Description 6 : SDS-PAGE Separating gel Component % of gel Acrylamide/Bis (40% w/v) 1.67mL 6.66mL 1.5M Tris-HCl (pH 8.8) 2.5mL 2.5mL Water .67mL 0.7mL TEMED |iL \iL % Ammonium persulphate 50 fiL 50 \xL SDS 100 £L 100 |iL For gradient gel 5-20% a gradient mixer connected to a peristaltic pump is used. Stacking gel (3%) Component mL Aery lamide/B is (40% w/v) 1.3 1M Tris-HCl (pH 6.8) 1.25 Water 7.4 TEMED 20 ^L % Ammonium persulphate 50 |uL SDS 100 jiL Running buffer 0.025M Tris, 0.192M glycine, 0.1% SDS, pH8.3 in water.

Sample buffer 1M Tris-HCl pH 6.8 13mL % SDS 6.5mL Glycerol 5.2mL 0.5%) Bromophenol blue 0.26mL Biorad Protean 2 equipment was used in accordance with manufacturer's instructions 76 Samples (total volume 15-20 )iL containing 1-10 jig sample protein) were loaded onto a gel.

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

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 Diphenvlchlorins and Bacteriochlorins - Their Synthesis and Bioconjugation for Targeted Photodynamic Therapy and Tumour Cell Imaging.J. Porphyrins and Phthalocyanines 4, 655-658; and Clarke, O.J. and Boyle, R.W. (1999) Isothiocvanatoporphyrins. useful intermediates for the conjugation of porphyrins with biomolecules and solid supports. JC.S. Chem. Commun. 2231-2232, each of which is incorporated herein by reference, that a set of porphyrin, 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 porphyrin 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 porphyrin-BSA conjugates into HeLa cells.

Our examples utilise 5,10,15-tris(3,5-dihydroxyphenyl)-20-(4-isothiocyanatophenyl) porphyrin (OH6) and 5,10,15-tris(pyridyl)-20-(4-isothiocyanatophenyl)porphyrin (PYR), as we have found from our previous studies that the pattern of hydrophilic substituents around the photoactive porphyrin core of each of these chromophores leads to efficient conjugation with proteins; hydrophilic substituents also minimise non-covalent binding of photosensitiser to protein, often found with more 77 hydrophobic porphyrins. Synthetic protocols for these chromophores are described in Examples 1 and 2 above respectively.

Example 25- stable conjugation to antibodies OH6 and PYR. were prepared as described in Examples 1 and 2 above respectively. Antibody 17.1 A was selected for the bioconjugation procedure. 17.1A is an antibody which reacts specifically with a receptor that is over-expressed on colorectal cancer cells, in particuiar 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 purposes 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.

Each of OH6 and PYR was separately 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..

HC HC (OH6) (PYR) 78 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. The results are shown in Figures 1-3 respectively. Figure 1 shows a gel loaded with buffer-exchanged 17.1 A antibody (lane 1), and buffer-exchanged antibody/OH6 conjugations at MRs 2.5 (lanes 2, 3), 5 (lanes 4, 5), 10 (lanes 6, 7) and 20 (lanes 8, 9) and molecular weight markers (lane 10). Figure 2 shows a gel loaded with dialysed 17.1 A antibody (lane 1), and dialysed antibody/OH6 conjugations at MRs 2.5 (lanes 2, 3), 5 (lanes 4, 5), 10 (lanes 6, 7) and 20 (lanes 8, 9) and molecular weight markers (lane 10). Figure 3 shows a gel loaded with buffer-exchanged 17.1A antibody (lane 1), and buffer-exchanged antibody/PYR conjugations at MRs 2.5 (lanes 2, 6), 5 (lanes 3,7), 10 (lanes 4, 8) and 20 (lanes 5, 9) and molecular weight markers (lane 10).

As seen in these Figures, neither the buffer-exchange nor dialysis procedures disrupt the antibody structure, the light and heavy chains remaining associated with one another and migrating together on each of the gels (lane 1). Conjugation of OH6 and PYR at each of the MRs can also be seen on the. gels (lanes 2-9).

Example 26 - FACS analysis FACS analyses were run in accordance with Methodology Description 5.

Figure 4 shows results derived utilising FITC-labelled 17.1A and Colo 320 cells (3 repeats) and indicates that binding of the antibody to the cells has occurred (ie the Colo 320 cells express the antigen specific to 17.1A).

Figure 5 shows results derived utilising OH6/17.1A conjugate and Colo 320 cells with a FITC-labelled anti-17.1A antibody for detection (3 repeats) and indicates that the OH6/17.1A conjugate has bound to the cells.

Figure 6 shows results derived utilising PYR/17.1A conjugate and Colo 320 cells with a FITC-labelled anti-17.1A antibody for detection (3 repeats) and indicates that the PYR/17.1A conjugate has bound to the cells.

Figure 7 shows results derived utilising FITC-labelled OX-34 which is an antibody of the same class (IgG2a) as 17.1A but with a different antigen specificity (3 repeats). The results indicate that OX-34 has not bound to the Colo 320 ceils and hence that there are no binding sites for OX-34 on Colo 320 cells.

Example 27 - Photocytotoxicity experiments Photocytotoxicity tests in accordance with the method described in Methodology Description 2 were performed on Colo 320 cells utilising various antibody conjugates.

Figures 8 and 9 show the results of control experiments performed using OH6/OX-34 and PYR/OX-34 conjugates respectively. 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.

Figures 10 and 11 show the results of further control experiments performed using "capped" OH6 and PYR respectively. The "capping" procedure involved reacting the NCS group on each chromophore with propylamine, so as to block serum protein conjugation. Figure 10 shows no cytotoxicity in the dark, indicating that OH6 is nontoxic to Colo 320 cells. On irradiation, however, some photocytotoxicity is observed, indicating that an amount of the capped OH6 has been transferred to the surface of the Colo 320 cells. Figure 11 meanwhile shows some cytotoxicity in the dark, suggesting that PYR is to some extent cytotoxic to Colo 320 cells, and increased photocytotoxicity on irradiation, which again indicates that an amount of the capped PYR has been transferred to the surface of the Colo 320 cells.

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

Figures 12 and 13 show results obtained using OH6/17.1A and PYR/17.1A conjugates respectively, at various conjugation dilutions (2.5, 5, 10, 20 for OH6/17.1A; 10 and 20 for PYR/17.1A). The results indicate a significant increase in cytotoxicity on irradiation, indicating that the binding of the bioconjugates to the cell surface confers photosensitivity upon the cells. Hence, these species are suitable candidates for PDT. 80 PCT/GBO1/02846 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. J. Cancer (1992) 65:813-817; RBoyle et al, Br. J. Cancer (1993) 67:1177-1181; RBoyle et al, Br. J. Cancer (1996) 73:49-53; and Lapointe et al, J. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876-882; the contents of each of which are incorporated 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, J. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876-882). As described in RBoyle et al, Br. J. 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 10,15,20-tris(3,5-dihydroxyphenyl)5-isothiocyanatophenylporphyrin (OH6-NCS) with BSA was carried 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 triplicate with sub-confluent cultures of HeLa cells, including a series of control solutions of unlabelled BSA, 10,15,20-tris(3,5-dihydroxyphenyl)5-aminophenylporphyrin porphyrin (OH6-NH2, amino precursor of OH6-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 Radiance2000 confocal laser scanning microscope (Bio-Rad Microscience, Cambridge, 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. Porphyrins 81 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-dimensionally by recording images from a series 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 |im to 1.25 )_im) 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: (i) a microscope slide containing medium, in order to measure background levels; (ii) a slide containing ITC porphyrin OH6-NCS dissolved in DMSO; and (iii) a slide bearing only un-probed HeLa cells.

Image data acquisition and remote microscope operation was carried out using the Bio-Rad Lasersharp2000 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 OH6-NCS at each of the excitation laser lines available on the CLSM set-up was carried out for a 0.01 mM solution of OH6 in DMSO. Figure 14 shows the UV-visible spectrum of OH6-NCS identifying its principal absorption bands. Unfortunately, no laser line was available in order to excite OH6-NCS at its Soret band Amax, Figure 15 demonstrates the relative intensities of fluorescence emission for OH6-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 OH6-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 absorption spectrum of OH6-NCS showed that the 516 nm argon-ion laser line was the only excitation source compatible with OH6-NCS. The three strongest laser lines, 457, 476, and 488 nm all excited in the region between the Soret and first Q band of OH6-NCS, whereas the 514 line overlapped well with the Q band at 516 nm. 82 Cell cultures separately incubated with conjugate OH6-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 OH6-NCS-BSA is shown in Figure 16 (this Figure should be viewed from top left to bottom right). Consecutive sections were scanned with a 2|_iM 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 OH6-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. The localisation of OH6-NH2 (unconjugated porphyrin control), is shown in Figure 17, which shows a CLSM image of porphyrin control cells with zoom view. No fluorescence was found to emanate from inside the cells, instead it appeared that the majority of OH6-NH2 had become localised on the plasma membrane. Evidently the BSA component of the conjugate is required in order to facilitate the transport of porphyrin to the interior of the cell.

In summary, it has been shown that the cellular localisation of porphyrin-BSA conjugates, constructed vm the formation of covalent thiourea linkages, can be imaged using conventional CLSM techniques. Unconjugated porphyrin OH6-NH2 was not found to penetrate the cellular membrane, whereas a significant level of fluorescence was detected from inside cells incubated with OH6-NCS-BSA, indicating good conjugate penetration. 83

Claims

Claims 84

1. A porphyrin chromophore of formula (I) below: IT A y "N21 22 N' 22 H V T N24 23 N no 12 10 or a chlorin chromophore of any of formulae (II), (III), (IV) or (V) below: 15 20 25 (II) (III) 30 II\ITCLL:ctual PkOr^TY OFFSCc OF N.Z. 2 7 MAY 2C34 RECSIVS9 85 10 or a bacteriochlorin chromophore of any of formulae (VI) and (VII) below: 15 (VI) wherein Ri is a phenyl group linked to a conjugating group Z; 20 Z is -NCS; 25 R.2 is pyridiniumyl or phenyl substituted by -0(Ci-C6 alkyl), pyridiniumyl or R4P+(R5)(R^)(R7); R4 is a single bond or C1-C6 alkyl; each of R5, R$ and R7 is independently hydrogen, C1-C6 alkyl or aryl optionally substituted by OH, C1-C6 alkyl, C1-C6 alkoxy, aryl, oxo, nitro, amino or cyano; R3 is hydrogen, CpC6 alkyl (which may optionally be substituted by one or more 30 halogen or OH groups) or an R2 group as defined above; 'NTtLL.VCTUAL n<OP£«TY OFFICE OF N.Z. 2 7 MAY 2m RECEIVED 86 each of X(, X2, X3 and X4 is independently selected from H, OH, halogen C1-C3 alkyl or -0(CrC3 alkyl); wherein each of Ri, R2 and R3 (when an R2 group) is optionally further substituted 5 with one or more hydrophilic substitutents selected from -OH, -CN, -NO2 or halogen.

2. A chromophore as claimed in claim 1, wherein R3 is H or C1-C6 alkyl substituted with one or more hydroxy groups. 10

3. A chromophore as claimed in claim 1, wherein R3 is an R2 group.

4. A chromophore as claimed in claim 3, wherein R2 and R3 are the same.

5. A chromophore as claimed in any one of claims 1 to 4, wherein R2 is: 15 ^ -W' P(W2)3 20 wherein Wi is Q-C6 alkyl; and W2 is hydrogen, C1-C6 alkyl or phenyl.

6. A chromophore as claimed any one of claims 1 to 5, wherein R2 is: 25 wherein W3 is hydrogen, hydroxy, methoxy or ethoxy. 30

7. A chromophore as claimed in any one of claims 1 to 6 wherein R2 is a phenyl ring with substituents in the meta- and/or para- positions. 2 7 may 26M D tc 87

8. A chromophore as claimed in claim 2 wherein R3 is: -CH2-CH(OH)-CH2-CH2OH.

9. A chromophore as claimed in claim 7 or claim 8, wherein R2 is a phenyl • 10 15 20 25 group substituted with methoxy, ethoxy, N-methylpyridiniumyl or methyltriphenylphosphonium.

10. A chromophore as claimed in any one of claims 1 to 9, wherein the conjugating group Z is at the para- position of the phenyl group Ri.

11. A chromophore as claimed in any one of claims 1 to 10, wherein the conjugating group Z is conjugated to a binding protein which is adapted to bind specifically to a biological target; or is conjugated to a bridging polypeptide which is adapted to bind to a complementary bridging polypeptide so as to couple the chromophore to said complementary bridging polypeptide.

12. A chromophore as claimed in claim 11, wherein the binding protein comprises an antibody.

13. A chromophore as claimed in claim 12, wherein the antibody is a monoclonal or polyclonal antibody or a fragment thereof with specificity for a target specific molecule on the surface of the biological target.

14. A chromophore as claimed in claim 12, wherein the antibody is a phage antibody, that is an antibody expressed on the surface of a bacteriophage.

15. A chromophore as claimed in claim 11 wherein the binding protein comprises a protein which is adapted to bind to one or more cell surface molecules or receptors; or a low density lipoprotein, which is adapted for insertion into a cell membrane. INTELLECTUAL PkOFc>*TY OFFICE OF HZ. 2 7 KAY 2EM RECEIVED 88

16. A chromophore as claimed in claim 15, wherein the binding protein is a serum albumin protein.

17. A chromophore as claimed in claim 11, wherein said bridging polypeptide comprises calmodulin and said complementary bridging polypeptide comprises calmodulin binding peptide; or vice versa; or said bridging polypeptide comprises . avidin or streptavidin and said complementary bridging polypeptide comprises biotin; or vice versa.

18. A chromophore as claimed in any one of claims 11 to 17, wherein said specific biological target is a cell or membrane, such as 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.

19. 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 one of claims 1 to 18, illuminating said mixture 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 the mixture of cells through a polarised environment so as to cause or allow said charged cells to be separated from said mixture.

20. A chromophore as claimed in any one of claims 1 to 18 for use in 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, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis or multiple sclerosis.

21. The use of a chromophore as claimed in any one of claims 1 to 18 in the preparation of a medicament for the treatment of a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as iNTELLCCTUAL PkOPcRTY OFFICE OF N.Z. 2 7 MAY 2C01! RECEIVED 89 tumours, cancers, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis or multiple sclerosis.

22. A pharmaceutical composition comprising a chromophore as claimed in any one of claims 1 to 18 and a suitable carrier.

23. A porphyrin chromophore according to any one of claims 1 to 18, or claim 20, substantially as herein described with reference to any one of examples 1 to 29, the. methodology description 1 to 6 and the figures 1 to 17 thereof.

24. A method according to claim 19, substantially as herein described with reference to the examples 1 to 29, the methodology description 1 to 6 and the figures 1 to 17 thereof.

25. A pharmaceutical composition according to claim 22, substantially as herein described with reference to the examples 1 to 29, the methodology description 1 to 26 and the accompanying figures thereof END OF CLAIMS "NiTtL l.-CTUAL PRO."I.iiY OFrSC? Op M 7. 2 7 MAY 2004 RECEIVED