Agr. Biol. Chem., 39 (9), 1781-1788,1975

The Degradation of Isopropylbenzene and Isobutylbenze ne by Pseudomonas sp.

Yoshifumi JIGAMI,Toshio OMORIand Yasuji MINODA Departmentof AgriculturalChemistry, Faculty of Agriculture, The Universityof Tokyo,Tokyo ReceivedMarch 17, 1975

To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107BI were examined. Oxidation products from isopropyl benzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyl-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3- isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains. From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropyl benzene and isobutylbenzene by these strains were discussed.

In the previous paper," the authors de convexa S107B1 described in the previous paper's were scribed the isolation of isopropylbenzene used for study. assimilation bacteria and the identification of Cultural methods. The composition of the medium the isolated strains, S107B1 and S182B1. and the culture conditions used for isolation of pro Furthermore, the substrate specificity differ ducts were the same as those reported for the microbial ence between bacteria assimilating various oxidation of ƒ¿-methylstyrene and ƒÀ-methylstyrene.2) aromatic hydrocarbons was reported. To Chemical. Isopropylbenzene and isobutylbenzene examine biodegradation pathways of these were obtained from Tokyo Chemical Industry Co., Ltd. aromatic hydrocarbons and the effect of 3-Isopropylcatechol was purchased from Aldrich Chemical Co., Inc. U.S.A. substituent groups of substrates on decom

posing pathways by microorganisms, the Analytical methods. Melting points were deter authors tried to isolate the intermediates from mined on a microscope hot stage and were reported culture broths. The effort resulted in the uncorrected. Optical rotations were measured in a JASCO DIP-S polarimeter. IR spectra were obtained isolation of new ring fission products from with a JASCO IR-S spectrometer. NMR spectra isopropylbenzene and isobutylbenzene by were recorded on a JEOL-JNM-4H spectrometer at

Pseudomonas species. This paper describes 100 MHz in CDCl3 with tetramethylsilane as an

the physical and chemical properties of these internal standard. Mass spectra were measured products and their chemical structures. From with a Hitachi RMU-6L mass spectrometer operating with an ionization energy of 70 eV. UV spectra were these results, the existence of an unknown measured on a Hitachi Model 124 spectrophotometer. reductive step in the degradation of these TLC were carried out with silica gel plates of 0.25 mm aromatic hydrocarbons was revealed. Fur thickness (Tokyo Kasei chromatogram sheet, type

thermore, both the initial attack on these S073), which were developed with the solvent system isoalkyl substituted aromatic hydrocarbons by of benzene-dioxane-acetic acid (90: 25: 4, v/v). Spots the strains and their metabolic pathways were were detected by spraying the following reagents: bromocresol green for acidic compounds, diazotized discussed. benzidine for phenolic compounds and 2,4-dinitro-

phenylhydrazine-HCl for keto compounds. MATERIALS AND METHODS Microorganisms. Ps. desmolytica S449B1 and Ps. 1782 Y . JIGAMI, T. OMORI and Y. MINODA

cultivated in 30 liter jar fermentors containing RESULT 20 liters of liquid medium and 100 ml of iso propylbenzene by the same way mentioned S1. Oxidation products from isopropylbenzene previously.' After cultivation, 80 liters of In order to examine oxidation products culture broth were collected, centrifuged to from isopropylbenzene, I liter of culture broth remove the cells and concentrated in vacuo to of strain S107B1 and S449B1 grown on is 8 liters. The concentrated culture broth was opropylbenzene for 2 days was fractionated and extracted with ether and fractionated accord extracted with ether to obtain an acidic frac ing to the procedures shown in Fig. 1. tion by usual methods . The results by TLC From the weakly acidic fraction, a crude showed that the main products differed be Product 1 was separated by silica gel column tween strain S107B1 and S449B1. On the chromatography using benzene as solvent. chromatogram, Ps. desmolytica S449B1 show Each eluate (5ml) was applied to TLC. The ed two dominant spots on Rf 0.71 and Rf zone at Rf 0.71 was cut, removed and ex 0.38, while Ps . convexa S107BI showed the tracted with ether. Evaporation of the solvent main spot on Rf 0.38. By spraying reagents, gave a brown oil (220 mg). Product I showed the spot on Rf 0.71 (Product 1) was known to positive coloration to diazotized benzidine be a phenolic compound and the spot on Rf and negative coloration to bromocresol green 0.38 (Product 2) was known to be an acidic and 2,4-dinitrophenylhydrazine. Judging from compound with a carbonyl group . For isola the coloration of dark brown to diazotized tion of these products, strain S449B1 was benzidine, Product 1 was assumed to be a

FIG. 1. Isolation Procedure for Product from Isopropylbenzene and Isobutylbenzene . Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas 1783

diphenolic compound. The IR spectrum TABLE 1. PHYSICAL AND CHEMICAL PROPERTIES OF PRODUCT 2 (film, CHCl3 sol.) showed absorption peaks at 3480cm-1 (hydroxyl), 1380cm-1 and 1360 cm-1 (isopropyl), 780cm-1 and 730cm-1

(aromatic). The mass spectrum revealed a molecular ion peak at m/e 152 and the fragmentation exhibited prominent ion peaks

at m/e 137 (M+-CH,), 123, 91, 77, 65, 51 and 43. From these data, Product 1 was

assumed to be a diphenolic compound with

an isopropyl group. Accordingly, in order to examine the presence of ortho dihydroxyl

groups on benzene nucleus, the formation of insoluble lead compound was checked by the method of Evans.3) In this test, Product 1

in neutral solution with lead acetate gave a dark green insoluble lead salt. Thus, the

presence of ortho dihydroxyl group was known. Furthermore, the NMR spectrum confirmed the structure of Product 1, including a sub

stitution of aromatic protons. Signals were at 82•`83•Ž, was positive to bromocresol observed at ƒÂCDCl3Me4Si 1.18 (6H, doublet, J= green and 2,4-dinitrophenylhydrazine and nega tive to diazotized benzidine. The IR spec 6.5Hz, isopropyl methyl), 3.16 (1H, multiplet, isopropyl methine), 5.42 (2H, singlet, hydroxyl, trum (in Nujol) showed the presence of hydro xyl group (3400 cm 1), carbonyl group (1700 disappeared on shaking with D,O) and 6.50- 6.80 (3H, multiplet, aromatic). In this case, cm-1) and isopropyl group (1378cm-1 and the entire spectrum, especially a chemical 1170cm-1). In the mass spectrum, a mole cular ion peak was not observed and the shift and a spectral pattern of aromatic fragmentation exhibited prominent ion peaks protons, was identical with authentic 3-iso at m/e 170 (M+-H,O), 145 (M+-C,H,), 99 propylcatechol. Thus, the structure of Pro duct 1 was determined to be 3-isopropylcate (C3H7COC2H5+), 85 (C3H7COCH2+), 71

chol. (C3H7CO+) and 57. On treatment with acetic anhydride-pyridine, Product 2 formed a mono On the other hand, for isolation of Product acetylated compound whose mass spectrum 2, a strongly acidic fraction was applied to showed a molecular ion peak at m/e 230. silica gel column chromatography using chloro Accordingly, the molecular weight of Product form as solvent. Each eluate (10g) which was 2 was determined to be 188, which was in passed through a silica gel column (3 x 40cm) was applied to TLC and fractions which gave good agreement with the result of elementary analysis (C9H16O4). The transparence in the a single spot of Rf 0.38 were collected. UV region suggested the absence of a con Evaporation of the solvent gave a crude

Product 2, which was dissolved in chloroform jugated double bond. Further, the NMR spectrum exhibited signals at ƒÂCDCl3Me4Si1.05 (6H, and passed through a silica gel column (I x doublet, J=7.0 Hz, isopropyl methyl), 1.82 20cm). Removal of the solvent after column chromatography resulted in the isolation of a (6H, multiplet, three methylenes), 1.90-.2.20 (2H, overlap of singlet hydroxyl and multiplet pale yellow crystal. Recrystallization from chloroform gave 1.83 g of colorless Product 2. isopropyl methine), 4.50 (1H, multiplet, hydro xy methine) and 4.90 5.20 (1H, broad singlet, Physical and chemical properties of Product 2 carboxylic). In this spectrum, however, the are summarized in Table I. Product 2 melted 1784 Y. JIGAMI, T. OMORI and Y. MINODA

FIG. 2. NMR Spectrum of Acetylated Product 2 (in CDCl3). spectral patterns of signals at b 1.90 - 2.20 and triplet of methylene protons at b 2.52 (J= b 4.50 were not clear and the assignment was 7.0Hz). Considering the chemical shift and difficult. spectral pattern, the triplet at b 2.52 was To further confirm the structure, the NMR recognized as a methylene group adjacent to spectrum of acetylated Product 2 shown in both methylene and oxo groups and the Fig. 2 was analysed. Signals were observed multiplet at b 2.60 was recognized as a at brasi 1.09 (6H, doublet, J=7.0 Hz, iso methine group adjacent to both isopropyl propyl methyl), 1.50-2.00 (4H, multiplet, methyl and oxo groups. Accordingly, the two methylenes),2.12 (3H, singlet, acetate partial structures of (CH3)2CHCOCH2CH2- methyl), 2.40-2.80 (3H, overlap of triplet and -CH2CH(OH)- were clarified, which were methyleneand multipletisopropyl methine), also supported by the fragmentation of mass 5.00 (1H, triplet, J=6.5 Hz, acetoxymethine) spectrum. Thus, the structure of Product 2 and 9.45 (1H, singlet,carboxylic, not shown was determined to be 2-hydroxy-7-methyl-6- in Fig. 2). In this spectrum, the spectral oxooctanoic acid. Product 2 indicated an pattern of the signalat b 5.00 due to methine optical activity of [a]D=+5.3° (c=0.16, proton was a typical triplet (J=6.5 Hz). CHCl3). Thus, the formation of (+)-2- Further, spin decouplingby irradiation at hydroxy-7-methyl-6-oxooctanoic acid from iso 5 1.90 due to methyleneprotons resulted in propylbenzene by Ps. desmolytica S449B1 was a marked change of pattern at b 5.00 from made clear. Product 2 was also isolated from triplet to ringlet. Thus, the triplet at b 5.00 the culture broth of Ps. convexa S107B1. was recognizedas an acetoxymethine group adjacentto methylenegroup. The signalsat 2. Oxidation products from isobutylbenzene b 2.40-2.80 was also clarifiedby the spin Strain S449B1 and S107B1 are known to decoupling. Irradiationat b 1.09due to iso grow on isobutylbenzene, a homologue of iso propyl methineproton resulted in a marked propylbenzene, as reported in the previous change of pattern at b 2.60 from multiplet paper." To examine oxidation products, the to singlet and no change was present in the crude acidic fraction obtained from 1 liter of Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas 1785 culture broth of strains grown on isobutyl group was known. The mass spectrum re benzene for 2 days was applied to TLC. vealed a molecular ion peak at m/e 166 and On TLC, a similar chromatogram to oxida prominent ion peaks were present at m/e 125 tion products of isopropylbenzene was found. (M+-C3H7), 105, 91, 77, 65 and 51. These That is, strain S449B1 showed two dominant properties were very similar to those of Pro- spots on Rf 0.73 (Product 3) and Rf 0.40 duct 1, and Product 3 was assumed to be an (Product 4), while strain S107B1 showed a ortho diphenolic compound with an isobutyl main spot on Rf 0.40 (Product 4). For iso group. Furthermore, the NMR spectral data lation of products, 80 liters of culture broth shown in Table II confirmed Product 3 to be of strain S449B1 grown on isobutylbenzene 3-isobutylcatechol. were obtained by the same way mentioned As for Product 4, the strongly acidic por previously and were separated according to tion was applied to silica gel column chromato the procedure shown in Fig. 1. graphy by the same way as Product 2 using From the weakly acidic portion, a crude chloroform as solvent. The second column Product 3 was obtained by silica gel column chromatography resulted in the isolation of a chromatography. After cutting out the zone pale yellow crystal, which was recrystallized at Rf 0.73 on TLC and extraction with from chloroform to leave 2.28 g of colorless ether, a brown oil (25mg) was isolated. Some Product 4. Physical and chemical properties properties of Product 3 are shown in Table II. of Product 4 are summarized in Table III. TABLEII. PHYSICALAND CHEMICAL TABLEIII. PHYSICALAND CHEMICAL PROPERTIESOFPRODUCT 3 PROPERTIESOFPRODUCT 4

The IR spectrum showed the presence of Product 3 was positive to diazotized benzidine hydroxyl group (3400cm-1), carbonyl group and negative to bromocresol green and 2,4- (1700cm-1) and isopropyl group (1366cm-1 dinitrophenylhydrazine. In the lead com- and 1170cm-1). In the MS spectrum, a mole pound formation test mentioned previously, cular ion peak was not observed and prominent Product 3 formed a dark green precipitate ion peaks were present at m/e 184 (M+-HBO), and the presence of ortho dihydroxyl phenolic 169 (M+-H,O-CH,), 141 (M+-H2O-C3H7), 1786 Y. JIGAMI,T. OMORIand ,;Y.!MINODA

FIG. 3. NMR Spectrum of Product 4 (in CDCl3).

127 (M+-C4H9), 113 (C4H9COC2H5+), 99 hydroxy methine proton. Accordingly, the

(C4H9COCH2+), 85 (C4H9CO+), 71, 57 and methine proton at 6 4.48 was assumed to be 43. These fragmentation patterns were quite connected with the carbon which was ad

similar to those of Product 2. Product 4 jacent to the carbon carrying methylene pro also formed a monoacetylated compound, tons at 3 1.85. However, in this spectrum, whose mass spectrum showed a molecular ion the spectral pattern of the signal at 3 4.48

peak at m/e 244. Accordingly, the molecular was not clear and the assignment was dif weight of Product 4 was known to be 202, ficult. To further confirm the structure, the

which was supported by the result of element NMR spectrum of acetylated Product 4 was ary analysis (C10H18O4). analysed. Signals were observed at

The NMR spectrum shown in Fig. 3ƒÂCDCl3Me4Si showed 0.91 (6H, doublet, J=7.0Hz, isopropyl me similar signals to those of Product 2, ƒÂCDCl3Me4Si thyl), 1.50-2.10 (5H, multiplet, overlap of 4 0.97 (6H, doublet, J=7.0 Hz, isopropyl two methylenes and an isopropyl methine), methyl), 1.60-2.20 (9H, multiplet, overlap of 2.13 (3H, singlet, acetate methyl), 2.30 (2H, four methylenes and an isopropyl methine), doublet, J=6.0 Hz, methylene), 2.45 (2H, 2.40-2.80 (2H, singlet, alcoholic and car triplet, J=7.0Hz, methylene), 5.00 (1H, tri boxylic, disappeared on shaking with D2O) plet, J=6.0Hz, acetoxy methine) and 9.43 and 4.48 (1H, multiplet, hydroxy methine). (1H, singlet, carboxylic). In this case, the In the spin decoupling experiment, a methine spectral pattern of the signal at 3 5.00 due to proton at 5 1.90 was known to be coupled methine proton was a typical triplet (J= with isopropyl methyl protons at o 0.97. 6.0Hz). A spin decoupling experiment Further, irradiation at 6 1.85 due to methylene showed that the acetoxy methine proton at protons sharpened the band at 6 4.48 due to 8 5.00 was coupled with the methylene pro- Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas 1787

tons at 8 1.90 and the methine protons at 6 isopropylbenzene and the formation of 3- 2.05 was coupled with the isopropyl methyl isobutylcatechol and (+)-2-hydroxy-8-methyl- protons at 6 0.91. Thus, the presence of 6-oxononanoic acid from isobutylbenzene by partial structures of (CH3)2CHCH2CO- and the same Pseudomonas strains mentioned -CH2CH(OH)- was known. Further, based above. These data showed that the initial on MS and NMR data which were quite oxidation of these isoalkyl substituted aroma analogous to those of Product 2, Product 4 tic hydrocarbons by these strains occurred on was determined to be 2-hydroxy-8-methyl-6- the benzene nucleus and not on the side oxononanoic acid, as shown in Fig. 3. Pro- chain of isopropyl or isobutyl group. duct 4 showed an optical activity of [a]01= As for oxidation products from isoalkyl -{-13.2° (c=0.10, CHCl3). Strain S107B1 substituted aromatic hydrocarbons, only the also formed Product 4. Thus, the formation paper by Baggi et a1.9) is available to the of (+)-2-hydroxy-8-methyl-6-oxononanoic acid best of our knowledge. They describe the from isobutylbenzene by Pseudomonas sp. was formation of cyclohexadienediols from 2- confirmed. phenylbutane and 3-phenylpentane and the formation of a meta ring fission product which DISCUSSION contains conjugated double bonds in its mole cule from 3-phenylpentane. Their results in Several studies have already been reported by dicated the initial oxidation also occurred on the authors on oxidation products of several the benzene nucleus and not on the isoalkyl n-alkyl substituted aromatic hydrocarbons side chain, which was the same result we by microorganisms.'" The authors recently obtained. However, for ring fission products, described the formation of a new ring fission our results were not analogous to those of product from n-butylbenzene by Ps. desmoly Baggi et al. Ring fission products formed tica and Ps. convexa.8) In the present paper, from isopropylbenzene and isobutylbenzene microbial degradation of iso-alkyl substituted were saturated compounds and contain no aromatic hydrocarbons was investigated to double bond. It was the same result with elucidate the metabolic pathways of these oxidation products from n-butylbenzene al compounds. The results indicated the for ready reported." Thus, the existence of the mation of 3-isopropylcatechol and (+)-2- unknown reductive step to form a saturated hydroxy - 7 - methyl- 6 -oxooctanoic acid from ring fission product mentioned above was gen-

FIG. 4. Proposed Pathways of Isopropylbenzene and Isobutylbenzene Degradation by Ps. desmoly tica and Ps. convexa. 1788 Y. JIGAMI, T. OMORI and Y. MINODA erally recognized in the degradation of these metabolic pathways involving them will be aromatic hydrocarbons by these strains. described elsewhere. Based on our knowledge and considering the ring fission product reported by Baggi REFERENCES et al., the decomposing pathways of isopropyl 1) T. Omori, Y. Jigami and Y. Minoda, Agr. Biol benzene and isobutylbenzene by Ps. desmoly . Chem., 39,1775 (1975). tica and Ps. convexa were proposed as shown 2) T. Omori, Y. Jigami and Y. Minoda, ibid., 38, in Fig. 4 which involves formation of the 409 (1974). new ring fission products, (+)-2-hydroxy-7- 3) W. C. Evans, Biochem. J., 41, 373 (1947). methyl-6-oxooctanoic acid and (+)-2-hydroxy- 4) T. Omori, S. Horiguchi and K. Yamada, Agr. Biol. Chem., 31,1337 (1967). 8-methyl-6-oxononanoic acid. As for the under 5) T. Omori and K. Yamada, ibid., 33, 979 (1969). metabolic pathways of these compounds, au 6) Y. Jigami, T. Omori, Y. Minoda and K. Yamada, thors were already successful in isolating ibid., 38, 401 (1974). further metabolites and are sure of the ex 7) Y. Jigami, T. Omori, Y. Minoda and K. Yamada, istence of another metabolic pathways of these ibid., 38, 467 (1974). 8) Y. Jigami, T. Omori and Y. Minoda, ibid., 38, aromatic hydrocarbons. But, further informa 1757 (1974). tion concerning the physical and chemical pro 9) G. Baggi, D. Catelani, E. Galli and V. Treccani, perties of the isolated compounds and the Biochem. J., 126, 1091 (1972).