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Affinity chromatography : variations on a heme

University of British Columbia

The development of a new affinity chromatography system for porphyrin and heme binding proteins is reported. The ligand was based on a protoporphyrin IX derivative substituted on either the 2- or 4-vinyl group. [Chemical Diagram] I:R₁=R₂=Vinyl,R₃H II:R₁=CHO,R₂=Vinyl,R₃=tBu III:R₁=Vinyl,R₂=CHO,R₃=tBu IV:R₁=-C=C-COOH,R₂=Vinyl,R₃=tBu V:R₁Vinyl, R₂=-C=C-COOH, R₃=tBu VI:R₁ =-C=C-CONH (CH₂ )₃NH₂,R₂=Vinyl,R₃=H VII:R₁=Vinyl,R₂=-C=C-CONH(CH₂)₆NH₂R₂K=Vinyl,R₃=H VIII:R₁=-C=C-CONH(CH₂ )₆NH₂, R₂=Vinyl, R₃=H IX:R₁=Vinyl; R₂=-C=C-CONH (CH₂)₆NH₂,R₃=H The ligand was synthesised from protoporphyrin IX (I) by oxidation to the monoformyl monovinyl derivatives (II and III) followed by a Knoevenagel condensation with malonic acid to the monoacrylic acid derivatives (IV and V). The aliphatic diamine was attached to the acrylic acid porphyrin through an acid chloride mediated amide linkage. The ligands VI, VII, VIII and IX were prepared by using propane 1,3— diamine or hexane 1,3-diamine respectively. The separation of the 2- and 4- isomeric porphyrins was carried out by employing the intermediacy of photoprotoporphyrin IX in the formation of monoformyl monovinyl deuteroporphyrin IX (II and III). This gave rise to the monoformyl porphyrin isomers (II or III) on a preparative scale and although not used in the later stages of the affinity ligand synthesis, the establishment of a reliable separation technique enhanced the potential selectivity and hence extended the general applicability of the affinity ligand. The porphyrin affinity ligand was coupled to cyanogen bromide activated sepharose to give a gel with a ligand concentration that could be varied from 4.7 μmoles/cm³ to 0.1 μmoles/cm³ by varying the activation procedure. Incorporation of iron into the porphyrin affinity ligand gave rise to a heme based affinity ligand. This in turn was coupled to cyanogen bromide activated sepharose for future use with the heme binding enzyme hemopexin. Attempts were made to form a porphyrinogen affinity ligand by reduction of the porphyrin with sodium borohydride but were unsuccessful. Initial affinity chromatography results with the porphyrin binding enzyme ferrochelatase are promising as binding onto the column has been definitely shown to occur. The potential synthetic usefulness of the intermediate monovinyl monoacrylic acid deuteroporphyrin is also discussed.

Text

2010-02-11

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http://hdl.handle.net/2429/20063

Chemistry

University of British Columbia

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