Polymer Journal, Vol. 23, No. I, pp 65-68 (1991)

NOTES Enantioselective Oxidation Catalyzed by Manganese(l11)­ Tetraphenylporphyrin-Bovine Serum Albumin Systems Modified or Unmodified with Poly(ethylene glycol)

Kohji YosHINAGA,* Naoki !TOH, and Taketoshi KITO

Department of Applied Chemistry, Kyushu Institute of Technology, Sensui, Tobata-ku, Kitakyushu 804, Japan

(Received June 27, 1990)

KEY WORDS Protein Hybrid / Metalloporphyrin / Bovine Serum Albumin / Asymmetric Oxidation/ Poly(ethylene glycol)/ Chemical Modification/

Protein hybrid recently becomes of much EXPERIMENTAL interest in therapeutics,1 enzymology,2 and biotechnology. 3 In our previous works,4 ' 5 we Materials have coupled the manganese(III)-tetraphen­ BSA (Fraction V) and poly(ethylene glycol) ylporphyrin complex to bovine serum albu­ monomethyl ether of molecular weight of 5000 min (BSA), which provides a chiral reaction. were purchased from Sigma Chemical and field, in order to approach to an artificial Aldrich Chemical Co., respectively. Complexes tryptophan dioxygenase, and shown that the of 5, 10, 15, 20-tetraphenylporphyrin manga­ hybrid catalytic system has exhibited the nese (III) chloride (MnCITPP) and 5, 10, 15, activity for stereoselective dioxygenation of 20-tetrakis (p-carboxyphenyl)porphyrin man­ methyl N-acetyl-L-( or D-)tryptophanate with ganese (III) chloride (MnCITCPP) were pre­ molecular oxygen, involving the indole-ring pared by the literature method. 7 Thioacetic cleavage. Moreover, it was found out that acid esters were synthesized by the method of attachment of poly(ethylene glycol) (PEG) to Newell et al. 8 the BSA hybrid catalyst prevented from deformation even in aqueous solvent contain­ Preparation of MnC!TCPP-BSA ing 60 vol¾ of tetrahydrofuran (THF). 5 MnClTCPP N-hydroxysuccinimidyl mono­ On the other hand, metalloporphyrins, ester was prepared from equimolar N­ widely studied as models of hemes or cyto­ hydroxysuccinimide with the manganese com­ chrome P-450, have been also known to ex­ plex using dicyclohexylcarbodiimide in N,N­ hibit the catalytic activity for monooxygena­ dimethylformamide. A mixture of the monoes­ tion, proceeding via the formation of high ter (17 mg, 0.02 mmol) and BSA (0.345 g, valency metal-oxygen complex intermediate. 6 5 x 10- 3 mmol) in pH 9 .3 buffer (0.05 moll - 1 In this work, we have examined asymmetric Na2 B4 0 7) was stirred at 4°C over day. epoxidation of olefins and oxidation of sufides Unbound manganese complex was removed by by the Mn(III) ClTPP-BSA hybrid catalytic a gel filtration (Sephadex G-50), followed by systems, and effects of PEG modification on a ultrafiltration (Toyo Roshi UK-50) with 0.05 their activities and enantioselectivities. mo11- 1 Na2 B4 0 7 solution containing 0.01

* To whom all correspondance should be addressed.

65 K. YOSHINAGA, N. {TOH, and T. KITO

1 mo11- NaN3 . A unit gram of BSA loaded (2 ml) and the borate buffer (2 ml) containing 60mg(6.9 x 10- 2 mmol) ofMnClTCPP, which MnClTCPP-BSA (0.1 g) was charged in a flask was determined by the absorbance of Soret and stirred for 2 h at 25°C, and 6 ml of bands (c = 5.44 x 104 mo1- 1 l cm) at 467 nm hydrogen peroxide aqueous solution (31 % ) with spectrophotometry. and 2 ml of the phosphate buffer were added. After stirring at 25°C for 1 or 2 days, the PEG Attachment to MnClTCPP-BSA sulfoxide produced was separated from the To 10ml of the borate buffer solution con­ reaction mixture in a similar manner as taining approximate 100 mg of MnCITCPP­ described previously, except for the use of BSA was added 100mg of methoxypolyeth­ hexane and successively diethyl ether-hexane ylene glycolyl N-succinimidyl succinate, pre­ (80: 20 vol¾) as eluent in chromatography. pared by the method of Abuchowski et al., 9 and the mixture was kept stirring for 2 h at RESULTS AND DISCUSSION 4°C. Unreacted PEG was removed with the ultrafiltration. It was determined that PEG Catalytic oxidation or epoxidation of olefinic attached to 33% of amino groups (mainly compounds with metalloporphyrin complexes lysine residue) of BSA, by means of Habeeb's was usually carried out using a proper oxidant, method10 using PEG-attached BSA derived such as NaOCl or NaIO4 , in the presence of from the same amount of BSA and the surfactant as a phase transfer medium. 11 When activated PEG under the same condition as in a mixture of styrene and MnClTPP in CH2Cl 2 the preparation of MnClTCPP-BSA-PEG. or CHC1 3 was stirred together with the phosphate buffer (pH 7 .2) solution of BSA and Epoxidation of Olefins with NaOCl aqueous solution for 4 days, A typical run was carried out as follows. To optically active styrene oxide in enantiomeric a mixture of MnClTCPP-BSA (0.1 g) in the excess (e.e.) of 3.6-4.8%, was produced in borate buffer (2 ml) and styrene (1.5 g, yield of 80-85% accompanied with benzalde­

14mmol) in CHC13 (10ml) were added 15ml hyde formation in the range of 2-3% yield ofNaOCI aqueous solution (35%) and 3 ml of (Table I). The catalytic reaction in the absence phosphate buffer (pH 7 .2, 0.2 moll - 1 ). The of BSA afforded small amount of styrene oxide resulting solution was magnetically kept in yield of 3.5% for 1 day. Thus, BSA possibly stirring at 25°C for 2 days. Extraction with works as both roles of a phase transfer medium three portions of chloroform ( 50 ml each) and and a chiral reaction field, while the reaction evaporation gave the crude product, which was by MnClTPP/BSA catalyst (prepared in situ) purified by chromatography on silica gel with proceeded slower than that by MnClTPP in hexane and successively chloroform-hexane the presence of surfactant such as N,N,N• (50: 50 vol¾). After determination of styrene trimethylbenzylammonium bromide. It has oxide by gas chromatography, its chloroform been reported that the surfactant-mediated solution was subjected to optical rotation reaction is usually involved with the formation measurement. In the same procedure using of an ion pair (~N+c1O-) and with its racemic styrene oxide instead of styrene, it was movement from aqueous phase to organic confirmed that recovered styrene oxide in­ phase, followed by the production of Mn(O)­ cluded no optically active contaminants of BSA CITPP complex. 6 On the contrary in the or its decomposed products. present reaction system, BSA, mostly existing in aqueous phase in the reaction mixture, Oxidation of Sulfides might trap MnClTPP and the substrate on the A mixture of sulfide (0.8 g) in organic solvent interface of two phases, and then transfer them

66 Polym. J., Vol. 23, No. I, 1991 Enantioselective Oxidation by Mn(III) TPP-BSA-PEG

Table I. Epoxidation of styrene by in situ prepared enantioselectivity comes from difficulty of rigid MnClTPP/BSA (I), MnCITCPP-BSA (II) coordination of styrene to hydrophobic bind­ and MnCITCPP-BSA-PEG (III) ing sites of BSA, because of unfunctionalized at 25°C at pH 7 .2° substrate. Interestingly, THF-H20 solvent Org. solv. Time Yield [o:]53 e.e.b system gave slightly higher e.e. (8-12%) of

Cat. styrene oxide than CHC1 3 or CH2 Cl 2-H2 0 vol% day % deg % heterogeneous solvent system. Water-miscible

I' CHCl3 (33) 4 85 1.2 4.8 solvents sometimes remarkably deform protein I' CH2 Cl 2 (33) 4 79 0.9 3.6 due to removal of liganded water molecules 1a CH2Cl2 (33) 2 81 1.2 4.8 sustaining the secondary and tertiary struc­ I' THF (10) 85 2.4 9.6 ture. 12 Indeed, we have observed that BSA is 1• THF (10) 24 0 0 I' THF (17) 54 2.2 8.8 deformed in THF (30 vol%)-H2 0 system day IJf CHC1 3 (33) 4 87 1.2 4.8 by day and more quickly in the solvent 111 THF (10) 4 92 2.9 11.7 containing 60 vol¾ ofTHF hour by hour. 5 In mi CHCl3 (33) 4 92 1.0 4.0 the heterogeneous system, the BSA hybrid • Styrene, 1.5 g; NaOCI solution, 15 ml. catalyst exists in aqueous phase, so that the h Calculated from [o:]53 -24.89° (c 2.86 in CHCl3)13 conformation of BSA is considered to be for (R)-styrene oxide. retained. Therefore, following two explana­ ' From 30mg of MnCITPP and 30mg of BSA. tions can be considered concerning the solvent a From 30 mg of MnCITPP and 30 mg of BSA-PEG. • From 30 mg of MnCITPP in the absence of BSA. effects on the catalytic enantioselectivity: (1) r Consisted of 6 mg of MnCITCPP and 0.1 g of BSA. hydrophilic circumstance ofTHF-H2 0 system gives more rigid coordination of styrene to the to the aqueous phase to react with oc1-. binding site of BSA through hydrophobic Indeed, efficient and rapid inclusions of interaction, and (2) BSA conformation de­ MnClTPP and a hydrophobic compound, such formed in the aqueous solvent is favorable for as methyl N-acetyl-L-tryptophanate, into BSA the enantio-face selective coordination of in a few minutes after mixing were observed styrene. In this case, the former seems to be by absorbance changes at 468 nm (Soret bands) plausible. and B bands at 280 nm, respectively, on Another olefinic compounds besides styrene, spectrophotometric spectra. 4 such as trans-stilbene, cc-methylstyrene, ethyl Meanwhile; PEG modification of protein cinnamate, gave a few inseparable byproducts, sometimes improves solubility and/or affinity in although the formation of corresponding organic solvents, hence we have examined the oxides was recognized. reaction using the PEG-modified BSA. As We next examined asymmetric oxidation of shown in Table I, an increase of the reaction sulfides, which have polar groups to be ex­ rate due to PEG modification was observed, pected to take rigid coordination to the BSA but the enantioselectivity was not affected. hybrid catalytic system, using hydrogen These results suggest that PEG attached on peroxide as an oxidant. By preliminary ex­ BSA contributes to only the phase transfer periments, it was shown that comparatively process, not to the asymmetric induction strong oxidants, such as NaOCI and m-chlo­ process. roperbenzoic acid, were inadequate for this The MnClTCPP-BSA catalytic systems reaction, because of bringing about rapid modified and unmodified by PEG did not spontaneous oxidation of sulfides used. As seen substantially improve the asymmetric selectiv­ in Table II, the sulfoxides produced from ity as compared with the catalytic systems (I phenyl methyl sulfide and 1-(phenylthio )acetic in Table I) prepared in situ. Probably, the low acid esters showed extremely low optical

Polym. J., Vol. 23, No. 1, 1991 67 K. YOSHINAGA, N. !TOH, and T. KITO

Table II. Oxidation of sulfides (R 1 -@-S- observed that the oxidation catalyzed by

CH2 COOR2) by II and III at 25°C at pH 7.2" MnClTCPP-BSA in CHC1 3-H2O system was involved with spontaneous reaction in con­ Sulfide Org. solv. Time Yield [a]l,3 e.e. siderable extent; about 50% conversion under ------Cat. the condition shown in Table II. Also, the R' R2 vol¾ day % deg % reaction in the presence of only BSA in CHC1 3 H Me II (0) 1 68 -0.3 (33 vol%)-H2O for 2 days resulted in H Bu II (0) 2 37 1.5 formation of the sulfoxide in 0.3% e.e. and in 3.7 <5b Me Me II (0) 92 62% yield, which corresponds to about 70% II THF (17) 94 5.2 7b II CH3CN (17) 96 0.9 of the product yield in the MnCITCPP-BSA III THF (I 7) 81 4.1 <5b catalyzed reaction. Therefore, it is suggested <5b III CHC1 3 (17) 30 3.1 that the BSA hybrid catalysts participate in the 71 9.7 5.4c Me Ph II (0) 3 asymmetric induction during the oxidation, II THF (17) 3 15 10 5.5c

II CHCl3 (33) 2 89 9.1 5.oc and also e.e.'s of sulfoxides produced by net CHC1 3 (33)d 2 62 0.5 0.3' catalytic reaction are approximately two or III THF (I 7) 2 25 9.4 5.2' three times higher than values shown in Table III CHCl (33) 2 79 13 7.2' 3 II. Inhibition of the spontaneous oxidation • Sulfide, 0.8 g; hydrogen peroxide solution (31 % ), 6 ml. and details in the present reaction are now b Determined by 1H NMR with Eu(hfch as a shift under investigation. reagent. 14 c Calculated from [a]0 + 180.9° (c 1 in CHC1 3) for (R)-( + )-phenyl 1-(p-tolylsulfinyl)acetate. REFERENCES d In the presence of only BSA (0.1 g). I. Y. Ikada, Biomedica, 3, 1154 (1988). rotation, although asymmetric induction was 2. Y. Inada, K. Takahashi, T. Yoshimoto, Y. Kodera, recognized. The oxidation of 1-(p-tolylthio )­ A. Matsushima, and Y. Saito, Trends Biotechnol., 6, 131 (1988). acetate derivatives catalyzed by MnClTCPP­ 3. D. Fisher and L. A. Sutherland, "Separation Using BSA complexes gave the optically active Aqueous Phase Systems; Applications in Cell Biology sulfoxide in low but reproducible e.e. (5-7%). and Biotechnology," Plenum, London, 1989. 4. K. Ohkubo, H. Ishida, K. Yoshinaga, T. Sagawa, K. The polar solvent of CH3CN-H2O system Urabe, M. Moriyasu, and H. Sone, Polym. Prepr., effected on the reaction rate, but markedly Jpn., 39, 824 (1990). suppressed the enantioselectivity. The bulky 5. K. Ohkubo, H. Ishida, K. Y oshinaga, T. Sagawa, phenyl ester group retarded the reaction, as and K. Urabe, Polym. Prepr., Jpn., 39, 825 (1990). compared with the methyl group, but did not 6. R. H. Holm, Chem. Rev., 87, 1401 (1987). e.e. 7. A. Harriman and G. Porter, J. Chem. Soc .. Faraday unexpectedly bring about an increase of of Trans. 2, 75, 1532 (1979). the sulfoxide. It is also notable that the reaction 8. 0. Newell and P. K. Calaway, J. Am. Chem. Soc., in heterogeneous solvent system of CHC1 3- 69, 116 (1949). H2O proceeded faster than that in homogen­ 9. A. Abchowski, G. M. Kazo, C. R. Verhoest, Jr, T. Van Es., D. Kafkewitz, M. L. Nucci, A. T. Vian, eous THF-H2 O system, in opposite phenom­ and F. F. Davis, Cancer Biochem. Biophys., 7, 175 ena in the epoxidation of styrene. In this case, (1984). the retained conformation of BSA in the 10. A. F. S. A. Habeeb, Anal. Biochem., 14,328 (I 966). heterogeneous system might be favorable for 11. J. H. Ramsden, R. S. Drago, and R. Riley, J. Am. Chem. Soc., 111, 3958 (1989). inclusion of the sulfide of phenyl ester. The 12. M. Waks, Proteins, 1, 4 (1986). effectiveness of PEG-modification of BSA was 13. J. Biggs, N. B. Chapman, and V. Wray, J. Chem. realized in the reaction in THF-H2O (17 vol% Soc. B., 71 (1971). · THF); increased yield of the product from 15% 14. R. Annunziata, M. Cinquini, and F. Cozzi, J. Chem. for 3 days to 25% for 2 days. However, it was Soc., Perkin Trans. I, 1687 (1979).

68 Polym. J., Vol. 23, No. I, 1991