Models for the photosynthetic water oxidizing enzyme II. Synthesis of covalently linked β-cyclodextrin-hemin

Abstract The active center of the enzyme responsible for the water splitting reaction of photosystem II is believed to be a binuclear, or possibly a tetranuclear cluster of Mn(III) and Mn(IV) ions [1, 2]. In addition to the manganese cluster, a cytochrome- b is believed to play an important role in the oxidation of water by the native enzyme. According to a scheme proposed by Cramer, high-potential cytochrome- b 559 may function in a cycle around PSII contributing to water splitting [3]. Moreover, a heme-protein which binds two manganese ions has been isolated from spinach chloroplasts [4, 5]. We reported earlier the synthesis of a Mn(III) dimer β-cyclodextrin [6]. In this communication we report on our efforts to synthesis a binuclear Mn(III)–β-cyclodextrin complex with is covalently attached to hemin via an ester linkage to the propionic acid side chains. Experimental Preparation of the Acid Chloride of Hemin(I). Hemin (1 m M , 0.65 g) was reacted with oxalyl chloride 92.5 m M , 0.31 g) under Ar at 0 °C. The reaction mixture was stirred for 6 hr and then any excess of oxalyl chloride was removed under vacuum. Preparation of Hemin derivatized with β-Cyclodextrin(II) . The crude product obtained above was dissolved in freshly distilled pyridine (30 mL). β-cyclodextrin (I m M , 1.3 g) was added to this solution and the reaction mixture stirred at room temperature for 10 hr. Precipitation of product was induced by solvent stripping and cooling. It was washed with chloroform and isolated by column chromatography on CM cellulose. On elution with DMF: CHCl 3 (9:1 by volume), two bands are obtained. After solvent stripping the separated products were precipitated by the addition of acetone, and analysis for Fe was carried out. The slow moving fraction gave satisfactory Fe analysis, whereas the fast moving fraction did not show the presence of Fe. Results and Discussion The synthetic approach was to convert the propionic acid side chains of hemin to the acid chloride followed by reaction with the primary hydroxyls of β-CD (Scheme I). Oxalyl chloride is an efficient reagent for converting carboxylic acids to acid chlorides [7]. The acid chloride derivative was reacted with an equimolar amount of β-cyclodextrin. It has been shown previously that under these conditions it is the primary hydroxyl groups of cyclodextrin which are more reactive [9]. The whole reaction is summarized in Scheme I. Both fractions obtained from the cellulose column were soluble in DMF and DMSO. The solubility of the Fe containing fraction in these solvents was, however, greatly reduced compared to free β-CD. Both fractions were fairly soluble in water, too, indicating that they may be precipitating as hydrochloride salts. Because only the slow moving fraction showed the presence of a significant amount of Fe, further characterization was attempted only on this sample. The electronic spectrum of this compound and of hemin are shown in Fig. 1. The 570 nm and 600 nm bands of hemin are lost and in their place three new bands appear at 500 nm, 568 nm and 564 nm. These changes are consistent with a significant perturbation of the π-π* transitions by a nearby β-Cd. The infrared spectrum of the compound shows the presence of all the prominent bands due to cyclodextrin and the ester carbonyl stretch at 1715 cm −1 . PMR of the compound in DMSO-d 6 showed that the C 1 H resonance is shifted to 4.82 ppm from 5.17 ppm, for the free cyclodextrin [8]. The O 2 H frequency is found to be shifted to 3.84 ppm from 4.38. The O 6 H proton signal is not observed, probably due to broadening because of fast exchange between O 6 H and the H + in the solvent. The upfield shift of 0.35 ppm in the C 1 H resonance is similar to the shift observed when β-cyclodextrin is ‘capped’ with terephthalolyl chloride [9]. Of the seven primary hydroxly groups of β-CD, the two which are involved in ester formation, have not been established yet. Reaction of this derivative with Mn(II) in an attempt to isolate the manganese containing derivative is in progress. This is expected to yield the binuclear manganese adduct with a structure analogous to that reported for the free β-CD, Fig. 2 [6]. Conclusion We have synthesized a covalently attached pair of macrocyclic ligands suitable for the coordination of a pair of Mn(III) ions via di-μ-hydroxo dimer structure and within 12 A of the Fe(III) core of hemin. It will be interesting to compare the magnetic, spectroscopic and catalytic properties of this compound in connection with its similarity to the photosynthetic water oxidizing enzyme.