Propionic side chains

To set the stage for the crucial aza-Robinson annulation, a reaction in which the nucleophilic character of the newly introduced thiolactam function is expected to play an important role, it is necessary to manipulate the methyl propionate side chain in 19. To this end, alkaline hydrolysis of the methyl ester in 19, followed by treatment of the resulting carboxylic acid with isobutyl chlorofor-mate, provides a mixed anhydride. The latter substance is a reactive acylating agent that combines smoothly with diazomethane to give diazo ketone 12 (77 % overall yield from 19). 

Hemozoin, also known as malaria pigment, is, in teims of its chemical composition, identical to (3-hematin. Hemozoin is formed as a crystallization product of heme under the acidic conditions present in the food vacuole of malarial parasites. In the crystal, the heme molecules are linked into dimers through reciprocal iron-carboxylate bonds to one of the propionate side chains of each porphyrin. The dimers form chains linked by hydrogen bonds. 

Esterification of the propionic acid side chain at C-13 (ring C) with a methyl group catalyzed by S-adenosyl-L-methionine-magnesium protoporphyrin 0-meth-yltransferase yields protoporphyrin IX monomethyl ester (MPE), which originates protochlorophyllide by a P-oxidation and cyclization of the methylated propionic side chain. This molecule contains a fifth isocyclic ring (ring E), the cyclopentanone ring that characterizes aU chlorophylls. 

Heme x Cyto bg subunit helix A cys35 Sy bonded to carbon atom of the porphyrin s C=CH substituent (Sy-C = 1.78 A) HjO-Fe Fe axial H2O ligand hydrogen-bonded to propionate side chain of heme bn (0H2---0 = 3.50 A) and cyto be gly38 backbone amide N-H (OH2-N-H = 2.70 A) 2.53... 

Figure 3/ for example/ places the lanosterol so as the 3f hydroxyl polar group lies over the propionate side chains. To reduce the complexity of this picture one can now replace the lanosterol structure by a surface canopy to represent the extent of the hydrophobic substrate binding site. There is also the facility to code this surface to signify the electronic properties of the substrates such as their electron density/ electrostatic potential/ or HOMO/LUMO values. Theoretical work of this type is currently suggesting quite remarkable complementarity of electron properties between bound substrates and protein binding sites. (10). 

For example, Hayashi et al. (73) recently reported an enhancement of the peroxidase activity of Mb by modification of two heme-propionate side chains. They prepared an artificial hemin having two benzene moieties linked at each terminal carboxylate of the heme-propionates in protoheme IX, as shown in Fig. 18. The modified hemin was then inserted into the horse heart apoMb to yield a reconstituted Mb. The characterization of the reconstituted Mb was carried out by ultraviolet-visible (UV-vis), NMR, and electronspray ionization-mass spectrometry (ESI-MS). Particularly, the UV-vis spectrum of the reconstituted Mb is comparable with that observed for the native Mb, suggesting that the artificial hemin is located in the normal position of the heme pocket. 

The initial turnover number of the thioanisole oxidation by the reconstituted Mb is clearly larger than that observed for the native Mb, and the kcat/Km value is more than seven fold higher than that of the native protein. The epoxidation of styrene is also effectively catalyzed by the reconstituted Mb by 10-fold increase compared to native Mb. These findings indicate that the appropriate modification of the heme propionate side chains forms a substrate-binding domain that enhances the peroxidase and peroxygenase activities of Mb, as shown in Table VI. 

Another example, which demonstrates the modulation of the peroxidase activity of Mb by modification of the heme-propionate side chains, has been proposed by Casella and co-workers (75). They prepared a Mb reconstituted with peptide-linked hemin such as protohemin-L-arginyl-L-alanine or protohe-min-L-histidine methyl ester as shown in Fig. 20. The peroxidase activity toward small substrate oxidations by the reconstituted Mb slightly increased the fccat... 

A chromophore such as the quinone, ruthenium complex, C(,o. or viologen is covalently introduced at the terminal of the heme-propionate side chain(s) (94-97). For example, Hamachi et al. (98) appended Ru2+(bpy)3 (bpy = 2,2 -bipyridine) at one of the terminals of the heme-propionate (Fig. 26) and monitored the photoinduced electron transfer from the photoexcited ruthenium complex to the heme-iron in the protein. The reduction of the heme-iron was monitored by the formation of oxyferrous species under aerobic conditions, while the Ru(III) complex was reductively quenched by EDTA as a sacrificial reagent. In addition, when [Co(NH3)5Cl]2+ was added to the system instead of EDTA, the photoexcited ruthenium complex was oxidatively quenched by the cobalt complex, and then one electron is abstracted from the heme-iron(III) to reduce the ruthenium complex (99). As a result, the oxoferryl species was detected due to the deprotonation of the hydroxyiron(III)-porphyrin cation radical species. An extension of this work was the assembly of the Ru2+(bpy)3 complex with a catenane moiety including the cyclic bis(viologen)(100). In the supramolecular system, vectorial electron transfer was achieved with a long-lived charge separation species (f > 2 ms). 

As mentioned above, both the point-mutation on the distal side of the Mb and the modification of the heme-propionate side chains are effective to convert the Mb into peroxidase and peroxygenase. Thus, one can imagine that the combination of an amino acid mutation and a modified-heme reconstitution may more effectively allow us a new catalyst by oxygen storage. Recently, two examples, which demonstrated the hybrid modification of Mb, have been reported. One is the T67R/S92D Mb reconstituted with the modified hemin where a histidine is linked at the terminal of the heme-propionate side chain (108). The peroxidase activities toward p-hydroxyphenylpropionic acid and tyramine oxidations by the reconstituted mutant Mb are increased by 24- and 2.3-fold, respectively, based on the kciJKm value compared to those observed for the native Mb. 

---