Synthetic approaches for the conjugation of porphyrins and related macrocycles to peptides and proteins.

The association of photosensitisers to peptides and proteins is a recognised and successful method for enhancing the selectivity and efficacy of photodynamic treatment. The covalent attachment of porphyrins and related macrocycles to peptides and proteins can generate new phototoxic species that allow the concentration of the oxidative damage to the target area, thanks to their enhanced cellular uptake, favourable sub-cellular distribution, and ability to target receptors or enzymes over-expressed by a given tissue or cell. The need to exert control over the regioselectivity of the conjugation led to the exploration of a variety of chemistries; in some cases based on bioorthogonal ligations, in others exploring the reactivity of naturally occurring functional groups. In this review we place a major emphasis on the synthetic strategies adopted to generate such conjugates. All such strategies will be surveyed, together with the methods used to introduce or unmask the appropriate reactive functionalities both on the peptide moiety and the photosensitiser.

Photosensitiser-antibody conjugates for photodynamic therapy.

Research into targeting of photodynamic sensitisers to specific biological tissue has been an area of increasing interest over the last twenty years. A significant number of these investigations have involved the use of antibodies or antibody fragments, and the field of photosensitiser bioconjugation is now an established area in its own right. This review seeks to bring together the diverse range of methodology now available for the efficient attachment of photodynamic agents to antibodies. The chemistry involved to obtain the complementary functionality required, on both photosensitiser and antibody, for successful conjugation is also discussed.

Identification of methicillin-resistant Staphylococcus aureus-specific peptides for targeted photoantimicrobial chemotherapy.

The increasing prevalence of multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), necessitates development of alternative modes of bacterial targeting which are not hindered by antibiotic resistance and minimise collateral damage. To achieve this, the FliTrx™ bacterially-displayed peptide library was panned against MRSA and randomly selected clones (n = 20) were DNA sequenced. One selected peptide was synthesised as both cyclic and linear constructs. Binding of the cyclic construct was observed by flow cytometry against isolates of MRSA whilst the linear construct showed low affinity. Low reactivity was observed with other Staphylococcal sp., gram-negative bacteria and human keratinocytes. The selected peptide was also cloned in-frame, within the thioredoxin gene into the pPROTet.E 6xHN vector for protein expression. A porphyrin photosensitiser (5-(4-isothiocyanatophenyl)-10,15,20-tris(4-N-methylpyridiniumyl)porphyrin trichloride) was conjugated to the recombinant protein and the in vitro cytotoxic effect of the resulting bioconjugate was determined against MRSA and other non-specific bacterial and mammalian cell lines. Photoantimicrobial chemotherapy (PACT) using the bioconjugate showed a 66% reduction in MRSA growth in comparison with non-irradiated cells. This work demonstrates the potential to isolate peptides with binding specificity against MRSA that can be used for targeted PACT, providing an effective alternative to antibody targeting.

Site-specific and stoichiometric conjugation of cationic porphyrins to antiangiogenic monoclonal antibodies.

Synthesis of three new cationic thiol-reactive maleimide-porphyrin derivatives and their use in site-specific conjugation to monoclonal antibodies is reported. The selective reactivity toward thiols is demonstrated using competition experiments, where both thiols and amines are present. This selectivity was used to successfully achieve specific conjugation of two porphyrins to cysteine residues present in the antiangiogenic antibody L19, expressed in small immunoprotein (SIP) format. The effect of length and hydrophilicity of the linkage between porphyrin and maleimide was also investigated, and maximum photocytotoxicity was achieved with the longest and most hydrophilic chain. Immunoreactivity and in vitro photocytotoxicity for these well-characterized porphyrin-antibody conjugates are described.

A new synthetic approach to N-arylquinolino[2,3,4-at]porphyrins from beta-arylaminoporphyrins.

A new reaction leading to porphyrins bearing fused rings is described. Novel N-arylquinolino[2,3,4-at]porphyrins 2 were obtained by thermal oxidative cyclization of beta-arylaminoporphyrins 1. The starting beta-arylaminoporphyrins were prepared by two routes: (i) nucleophilic displacement of the nitro group from 2-nitro-5,10,15,20-tetraphenylporphyrin by anilines and (ii) palladium-catalyzed amination of bromobenzene derivatives with (2-amino-5,10,15,20-tetraphenylporphyrinato)nickel(II). The N-arylquinolino[2,3,4-at]porphyrins show interesting UV-vis spectra with strong absorption bands in the red region.

An easy synthetic approach to pyridoporphyrins by domino reactions.

Beta-amino-meso-tetraarylporphyrins react with cyclic enol ethers to yield pyrido[2,3-b]porphyrins bearing two vicinal hydroxyalkyl groups. These reactions are catalyzed by lanthanum triflate and occur under mild conditions. Esterification of the hydroxyalkyl groups with succinic anhydride and dodecanoyl chloride afforded the corresponding esters in almost quantitative yields. The crystal structure of the most hydrophobic derivative 7 was determined, and it shows that these porphyrinic macrocycles form one-dimensional supramolecular tapes in the solid state.

Synthesis of novel N-linked porphyrin-phthalocyanine dyads.

[structure: see text] Two types of covalently NH-linked porphyrin-phthalocyanine dyads, connected either through the meso phenyl group or the beta-pyrrolic position of the porphyrin, have been synthesized following statistical condensation methodologies for phthalocyanine preparation and palladium-catalyzed amination methods. Photophysical studies have revealed that energy transfer from the porphyrin to the phthalocyanine prevails regardless of linkage.

Unexpected fragmentation of beta-substituted meso-tetraphenylporphyrins induced by high-energy collisional activation.

The protonated molecules and radical cations of meso-tetraphenylporphyrins with beta-pyrrolic substituents, when formed by fast atom bombardment (FAB) and subjected to high-energy collisions, give rise to unexpected fragment ions. The reaction involves hydrogen migration from the ortho position of the phenyl ring to the a atom of the substituent, with formation of an intramolecular, six-membered ring. The process is analogous to condensed-phase cyclizations described for the same type of compounds. The fragmentation requires the presence of a double bond in the substituent group attached to the pyrrolic ring. A rearrangement process involving anchimeric assistance by the phenyl group (analogous to an ortho effect) is proposed for the formation of these ions.

Synthesis of neutral and cationic tripyridylporphyrin-D-galactose conjugates and the photoinactivation of HSV-1.

Neutral and cationic tripyridylporphyrin-D-galactose conjugates were synthesized and their antiviral activity against herpes simplex virus type 1 (HSV-1) was evaluated. At non-cytotoxic concentrations the studied compounds show significant antiviral activity after photoactivation. The influence of photoactivation on drug treated cells was also analyzed, at different times of infection with HSV-1, for a neutral (1b) and a cationic glycoporphyrin (3b) derivative. The results show that the inhibition of the viral yield is more dependent on photoactivation for compound 1b than for compound 3b. These two compounds also differ in the inhibitory effect during the viral replicative cycle: while compound 3b inhibits the viral yield at all the addition times assayed, compound 1b is more efficient in later times of infection.