APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1996, p. 3355–3359 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 62, No. 9
Regio- and Stereospecific Oxidation of 9,10-Dihydroanthracene and 9,10-Dihydrophenanthrene by Naphthalene Dioxygenase: Structure and Absolute Stereochemistry of Metabolites SOL M. RESNICK*
AND
DAVID T. GIBSON
The Department of Microbiology and The Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242 Received 29 March 1996/Accepted 21 June 1996
The oxidation of 9,10-dihydroanthracene and 9,10-dihydrophenanthrene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4. Salicylateinduced cells of P. putida strain 9816/11 and isopropylthiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized 9,10-dihydroanthracene to (1)-cis-(1R,2S)-1,2-dihydroxy-1,2,9,10-tetrahydroanthracene (>95% relative yield; >95% enantiomeric excess) as the major product. 9-Hydroxy-9,10dihydroanthracene (95% enantiomeric excess) and (1)-(S)-9-hydroxy-9,10-dihydrophenanthrene (30% relative yield). The major reaction catalyzed by naphthalene dioxygenase with 9,10-dihydroanthracene and 9,10dihydrophenanthrene was stereospecific dihydroxylation in which both of the previously undescribed cis-diene diols were of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The results suggest that for benzocylic substrates, the location of benzylic carbons influences the type of reaction(s) catalyzed by naphthalene dioxygenase. new applications and more diverse targets (18). Information about the regio- and stereospecific oxidations catalyzed by different bacterial dioxygenases may also assist in the rational prediction of chiral products obtainable through the microbial oxidation of prochiral substrates. Toluene dioxygenase (TDO) expressed by Pseudomonas putida UV4 catalyzes the benzylic monooxygenation of 9,10dihydroanthracene (DHA) to 9-hydroxy-9,10-DHA as the only product (6). Differences in the regioselectivity and stereospecificity of the reactions catalyzed by NDO and TDO (2, 13, 26, 31, 32, 35, 41, 42) have led us to examine the oxidation of the three-ring hydroaromatic substrates DHA and 9,10-dihydrophenanthrene (DHP) by NDO. On the basis of previous NDOcatalyzed oxidation reactions (13, 41), we reasoned that both DHA and DHP would undergo cis dihydroxylation and/or monohydroxylation and that DHP, which contains two adjacent methylenic carbons, may be a substrate for oxygen-dependent desaturation by NDO. We report here the oxidation of DHA and DHP to cis-diol and monol metabolites by strains expressing NDO from Pseudomonas sp. NCIB 9816-4.
Naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 is a multicomponent enzyme which catalyzes the enantiospecific incorporation of dioxygen into naphthalene to form (1)-cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (12, 21). In addition to catalyzing cis-dihydroxylation of naphthalene and related aromatic compounds (22–24), NDO also catalyzes monooxygenation (13, 35, 42), sulfoxidation (3, 26), O-dealkylation (31), and desaturation (13, 31, 41) reactions for a number of aromatic and benzocyclic compounds. It has been suggested that these reactions are catalyzed by the same strong oxidizing species formed from the interaction of oxygen with mononuclear iron in the large (a) subunit of the terminal oxygenase and that the diversity of reactions catalyzed is dependent on interactions between the active site of the oxygenase and the structure of the substrate (13). The growing demand for single-enantiomer forms of chiral pharmaceuticals coupled with efficient enantioselective synthesis of a wide variety of biologically active natural products has led to an interest in the production of chiral synthons by microbial biocatalysis (19, 39). Some of the most versatile synthons are the homochiral arene-cis-diols formed by the bacterial dioxygenation of mono- and polycyclic arenes (14, 15, 36). Owing to the high enantiomeric purity of bacterial cis-diols, biocatalytic approaches have been employed in numerous environmentally benign enantiocontrolled syntheses of novel acyclic sugars, conduritols, inositols, and complex bioactive natural products, often with superior yields and in fewer steps than by conventional synthetic routes (7, 8, 18, 19, 37). The identification of new chiral cis-diols from more complex and highly substituted compounds may lead synthetic chemists toward
MATERIALS AND METHODS Bacterial strains and growth. P. putida 9816/11 is a mutant derived from P. putida NCIB 9816-4 (23) which oxidizes naphthalene to homochiral (1)-cisnaphthalene dihydrodiol (41). Cultures of strain 9816/11 were grown in 800 ml of mineral salts base (MSB, pH 7.2) (38) containing 0.2% pyruvate (wt/vol). NDO was induced during the log phase of growth with 0.05% (wt/vol) salicylate (4), and the cells were harvested as described previously (31). Cultures of the wildtype strain NCIB 9816-4 were grown under identical conditions but were induced with 0.05% (wt/vol) anthranilate (4). Escherichia coli JM109(DE3)(pDTG141) contains the NDO structural genes (nahAaAbAcAd) for NDO on plasmid pT7-5, where expression is under control of an isopropylthiogalactopyranoside (IPTG)inducible lac promoter (40). Cells were grown as previously described (33) and were used immediately or stored at 2708C. E. coli JM109(DE3)(pT7-5) was used in control experiments. Biotransformation of DHA and DHP. Induced cells of strain 9816/11 were resuspended in 400 or 500 ml of 0.05 M sodium-potassium phosphate buffer (pH 7.3) (optical density at 600 nm of 2.0 to 3.2) and transferred to 2.8-liter Fernbach flasks containing 0.05% DHA (wt/vol) (predelivered from an acetone stock
* Corresponding author. Mailing address: The Department of Microbiology and The Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7982. Fax: (319) 335-9999. Electronic mail address:
[email protected]. 3355
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TABLE 1. Physical properties of products formed from 9,10-DHA and 9,10-DHP by strains expressing NDO Compound
Substrate
I (Diol)
DHA
II (Monol)
DHA
III (Diol)
DHP
IV (Monol)
DHP
[a]Dc (concn [g/ml])
H NMR dataa
GC-MS datab
Exact (calculated) mass
d 3.47 (m, 2H), 3.56 (dt, J 5 21.5 Hz, 5.1 Hz, 1H), 3.72 (dt, J 5 21.5 Hz, 5.5 Hz, 1H), 4.10 (d, J 5 6.1 Hz, 1H), 4.39 (dd, J 5 6.1 Hz, 2.1 Hz, 1H), 5.85 - 5.94 (m, 2H), 7.15 7.19 (m, 4H) NDd
(PBA) Rt 17.65 min; m/z 300 (79), 285 (7), 222 (5), 195 (14), 179 (41), 178 (100), 172 (12), 165 (36), 153 (19), 141 (14), 128 (19) Rt 8.51 min; m/z 196 (78), 195 (100), 178 (67), 165 (27), 153 (16), 89 (20), 76 (20)
300.1310 (300.1322)
d 2.30-2.43 (m, 2H), 2.77 - 2.83 (m, 2H), 4.56 (d, J 5 5.7 Hz, 1H), 4.58 - 4.61 (m, 1H), 5.87 (d, J 5 7.7 Hz, 1H), 5.94 (dd, J 5 9.5 Hz, 2.4 Hz, 1H), 7.14 - 7.25 (m, 3H), 7.48 (d, J 5 7.7 Hz, 1H) d 2.98 (dd, J 5 15.1 Hz, 8.1 Hz, 1H), 3.04 (dd, J 5 15.1 Hz, 4.8 Hz, 1H), 4.81 (dd, J 5 8.1 Hz, 4.8 Hz, 1H), 7.22 (td, J 5 7.4 Hz, 1.1 Hz, 1H), 7.25 - 7.32 (m, 3H), 7.36 (td, J 5 7.6 Hz, 1.4 Hz, 1H), 7.51 (d, J 5 7.4 Hz, 1H), 7.80 (d, J 5 7.7 Hz, 2H)
(PBA) Rt 17.07 min; m/z 300 (100), 285 (6), 272 (6), 196 (24), 178 (19), 167 (26), 165 (21), 153 (15)
300.1304 (300.1322)
1186 (3.5)
Rt 8.67 min; m/z 196 (100), 195 (54), 181 (25), 179 (32), 178 (57), 167 (32), 165 (42), 152 (28), 139 (6)
ND
127 (1.5)
1
ND
195 (0.7)
NA
a Determined in CDCl3 at 360 MHz (compounds I to III) or in d4-methanol at 600 MHz (compound IV). Chemical shift (d) multiplicities are abbreviated as follows: s, singlet; d, doublet; t, triplet, m, multiplet; dd, doublet of doublets; td, triplet of doublets; J, coupling constant. b Rt and ion fragmentation patterns (% relative intensity) were determined under the conditions described in the text. PBA indicates that the diols were analyzed (GC-MS and HR-MS) as their phenyl boronic acid derivatives. c Specific rotations were determined in methanol (258C) at the concentration listed (g/ml). NA, not applicable. d ND, not determined.
solution [33]) or 0.1% DHP (vol/vol). Pyruvate (0.2%) was provided as an energy source and Tween 80 (0.02%) was added to improve dispersion of substrates in the aqueous medium. Biotransformation reactions were incubated at 308C with shaking (220 rpm) for 20 h. Preliminary studies showed maximum product accumulation at this time. Excess DHA was removed by filtration through glass wool. Cells were removed by centrifugation, and compounds in the supernatants were isolated as described below. IPTG-induced cells of E. coli strains JM109(DE3)(pDTG141) and JM109(DE3)(pT7-5) were incubated as described above except that 0.5% pyruvate was provided as the energy source. Product isolation, identification, and analysis. Supernatants from biotransformation reactions were extracted with ethyl acetate and analyzed by thin-layer chromatography (TLC) (33). Following the extraction of neutral metabolites, supernatants from reactions with wild-type strain 9816-4 were acidified (pH 2.5, HCl) and extracted three times with ethyl acetate. The extracts were washed twice with 0.1 volume of water, dried over Na2SO4, and concentrated to dryness. Metabolites were isolated by preparative-layer chromatography (PLC) with chloroform-acetone (8:2) or radial-dispersion chromatography (RDC) (35). Specific rotations ([a]D), chiral stationary-phase high-performance liquid chromatography (CSP-HPLC), proton nuclear magnetic resonance spectrometry (1H NMR), and high-resolution mass spectrometry were performed as previously reported (33, 35). 1H NMR analysis at 600 MHz was performed on a Bruker AMX600 spectrometer. Gas chromatography-mass spectrometry (GC-MS) was performed as described previously (35) except that the temperatures of the inlet and transfer line were 250 and 2808C, respectively, and the oven was programmed from 150 to 2758C at 108C/min. The (1)-(S)- and (2)-(R)-enantiomers of 9-hydroxy-9,10DHP were resolved by CSP-HPLC (Chiralcel OJ column) at retention times of 22.5 and 23.7 min, respectively. Phenyl boronic acid (PBA) derivatives (16) were prepared by the addition of PBA (1 eq) to isolated cis-diols (1 to 2 mg) dissolved in 0.5 ml of acetone. Circular dichroism (CD) spectra were obtained at 258C in methanol with an Aviv model 62DS CD spectrometer (Lakewood, N.J.). The methanolic solutions had an absorbance of 1.0 at the wavelength of maximal absorption [lmax] in a 1.0-cm-path-length quartz cuvette in a UV-vis spectrophotometer. Diastereomeric boronate esters were prepared with (2)-(S)- and (1)-(R)-2(1-methoxyethyl)-phenyl boronic acid (MPBA) as described previously (34). The MPBA esters were filtered through small plugs of sodium sulfate with CHCl3, concentrated to dryness, and dissolved in 0.6 ml of CDCl3 (containing 0.05% tetramethylsilane as an internal reference; 0 ppm) for 1H NMR analysis. Chemicals. DHA, DHP, anthrone, and PBA were from Aldrich Chemical Co. (Milwaukee, Wis.). (2)-(S)- and (1)-(R)-MPBA were prepared as described previously (34). 9-Hydroxy-9,10-DHA was prepared by treatment of anthrone with sodium borohydride (4 eq) for 2 h in methanol. All other chemicals were commercially available and were used without further purification. Racemic 9-hydroxy-9,10-DHP (,1 mg) was prepared by incubating 400 ml of IPTGinduced E. coli JM109(DE3)(pT7-5) cells with DHP under the biotransformation
conditions described above. The compound was isolated from the ethyl acetate extract of the supernatant by PLC.
RESULTS AND DISCUSSION DHA biotransformation by strains expressing NDO. Induced cells of P. putida strain 9816/11 (23 400-ml reaction volumes) incubated with DHA gave 110 mg of crude products. TLC analysis showed a major UV-quenching product (compound I) with an Rf of 0.2. Compound I was isolated by PLC (1-mm silica, 76 mg applied) to yield 36 mg of a white solid with the characteristics listed in Table 1. The 1H NMR spectrum showed chemical shifts resulting from four aromatic protons, two diene protons (5.80 ppm), two spin-coupled hydroxymethine protons (4.10 and 4.39 ppm, coupling constant [J] 5 6.1 Hz), and four upfield methylenic protons which identified compound I as 1,2-dihydroxy-1,2,9,10-tetrahydroanthracene. A cis relative stereochemistry for the vicinal hydroxyl groups was inferred by the 1H NMR hydroxymethine (carbinol) coupling constants of J1,2 5 6.1 Hz (22) and by highresolution mass spectrometry analysis of the derivative formed with PBA which showed an exact mass of 300.1310 (C20H17O2B requires 300.1322) (Table 1). When analyzed by GC-MS, the underivatized cis-diol dehydrated to yield approximately equal amounts of 1- and 2-hydroxy-9,10-DHA (M1 196). IPTG-induced cells of E. coli JM109(DE3)(pDTG141) oxidized DHA to a major product (approximately 50 mg/liter) with the same GC-MS, NMR, and chiroptical properties as compound I formed by strain 9816/11. A minor product (compound II) formed from DHA by the mutant and recombinant strains expressing NDO was detected by GC-MS analysis and represented ,5% of the total ion current area (Table 1). Compound II had an identical retention time (Rt) and mass spectrum to that of authentic 9-hydroxy-9,10-DHA. DHA oxidation was not observed in control experiments with strain JM109(DE3)(pT7-5). DHA did not
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FIG. 2. Major products formed from (A) 9,10-DHA and (B) 9,10-DHP by strains expressing NDO. The minor product (,5% relative yield) formed from DHA (9-hydroxy-9,10-DHA; compound II) is not shown. The wild-type strain NCIB 9816-4 oxidized DHP to compound IV as the sole neutral product. Product yields are reported in the text.
FIG. 1. Expanded regions of the 1H NMR spectra showing the diastereomeric methoxy (OMe) and methyl (Me) signals resulting from the esters formed with (1)-cis-(1R,2S)-1,2-dihydroxy-1,2,9,10-tetrahydroanthracene (compound I, via strain 9816/11) and (2)-S-MPBA (d OMe 5 3.199 ppm; d Me 5 1.431 ppm) (A) and a mixture of (1)- and (2)-MPBA (B). The signals resulting from the OMe and Me groups of the diastereomeric ester formed with compound I and (1)-R-MPBA were observed at 3.234 and 1.395 ppm, respectively.
support growth of the wild-type strain NCIB 9816-4. However, induced cells of NCIB 9816-4 oxidized DHA to small amounts of compound II, as the sole neutral metabolite, and acidic products which were not identified. Absolute configuration of compound I. Compound I eluted as a single symmetrical peak at an Rt of 23.62 min when analyzed by CSP-HPLC (Chiralcel OB-H), and its enantiomeric purity was determined by 1H NMR analysis of the diastereomeric esters formed with (2)-(S)- and (1)-(R)-MPBA. The singlet resulting from the methoxyl group of the ester formed with compound I and (2)-(S)-MPBA (3.199 ppm) was shifted upfield of the corresponding signal given by the (1)-(R)MPBA ester (3.234 ppm) (Fig. 1). The observed directional shift was consistent with the trend observed for the MPBA esters prepared from a series of related cis-diols of (1R,2S)absolute stereochemistry (34). The 1H NMR spectrum of the ester formed with compound I and (2)-(S)-MPBA showed no detectable chemical shift at 3.234 ppm. On the basis of the above results, compound I was identified as (1)-cis-(1R,2S)1,2-dihydroxy-1,2,9,10-tetrahydroanthracene (.95% enantiomeric excess [ee]) (Fig. 2). The absolute stereochemistry of this new diol is consistent with that of other cis-diols formed by NDO. DHP biotransformation by strains expressing NDO. Induced cells of strain 9816/11 (500 ml) were incubated with
0.1% DHP as described in Materials and Methods. Ethyl acetate extraction and subsequent workup gave 130 mg of crude products. TLC analysis showed the presence of two major UV-quenching products with Rf values of 0.2 (compound III) and 0.5 (compound IV). Both compounds were isolated by RDC (2-mm silica) to yield approximately 50 mg of compound III and 20 mg of compound IV. Their physical properties are listed in Table 1. The same products were formed from DHP by IPTG-induced cells (500 ml) of E. coli JM109(DE3) (pDTG141). RDC of 102 mg of crude products yielded 36 mg of compound III and 15 mg of compound IV. The 1H NMR and mass spectra of compound III were consistent with the structure 3,4-dihydroxy-3,4,9,10-tetrahydrophenanthrene. The chemical shifts of the hydroxymethine protons (H-3 and H-4) at 4.58 and 4.56 ppm and their coupling constants of J3,4 5 5.7 Hz were similar to the values reported previously for the bay region cis-3,4-dihydrodiol of phenanthrene (22), and a cis relative stereochemistry of the hydroxyl groups is further supported by the high-resolution mass spectrum of its PBA derivative (Table 1). These properties and the specific rotation identify compound III as (1)-cis-3,4-dihydroxy-3,4,9,10-tetrahydrophenanthrene. GC-MS analysis of compound III (underivatized) resulted in the formation of phenolic dehydration products (M1 196). However, GC-MS analysis of the PBAderivatized material showed compound III (M1 300, Table 1) and small amounts of two compounds with M1 at m/z 298 and 300. The M1 at m/z 298 is consistent with the PBA derivative of cis-3,4-phenanthrene-3,4-dihydrodiol, and the latter M1 is consistent with a cis-tetrahydrodiol of unknown regiochemistry. These minor products were not isolated or further characterized. The putative cis-3,4-phenanthrene dihydrodiol could be formed by desaturation of DHP or dehydration of compound IV (see below) followed by cis dihydroxylation of phenanthrene. NDO has previously been shown to catalyze desaturation reactions with indan, 1,2-dihydronaphthalene, ethylbenzene, and phenetole (13, 27, 31, 41). Although enzymic alcohol dehydroxylation or dehydration can be catalyzed by anaerobic microorganisms (17), dioxygenase-catalyzed dehydroxylation reactions have not been observed to date.
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Compound IV was identified as an isomer of hydroxy-9,10DHP from its mass spectrum, which gave an M1 at m/z 196. Its 1 H NMR spectrum showed the presence of eight aromatic protons, two methylenic protons, and a single hydroxymethine (carbinol) proton at 4.81 ppm; these results established the position of the hydroxyl group at C-9 and identified compound IV as (1)-9-hydroxy-9,10-DHP (Table 1). Although this compound is stable with respect to acid-catalyzed dehydration (30), we observed the presence of phenanthrene (up to 15%) when purified compound IV was analyzed by GC-MS. This was presumably due to dehydration of the compound in the GC inlet. Control experiments containing DHP and E. coli JM109(DE3) (pT7-5) cells yielded a small amount of compound IV (identified by GC-MS). The isolated yield (0.6 mg/400-ml biotransformation) was approximately 5% of the yield of compound IV obtained with E. coli JM109(DE3)(pDTG141) expressing NDO. Subsequent CSP-HPLC analysis indicated that compound IV formed by strain JM109(DE3)(pT7-5) was racemic. DHP did not support growth of the wild-type strain NCIB 9816-4. However, induced cells of the wild-type strain NCIB 9816-4 oxidized DHP to 9-hydroxy-9,10-DHP (compound IV) as the only neutral product detected. RDC (1-mm silica) of the neutral extract (27 mg) from a 400-ml DHP biotransformation with induced cells of strain NCIB 9816-4 yielded 12 mg of 9-hydroxy-9,10-DHP. This was due to the fact that compound III was metabolized to acidic products by the enzymes responsible for the degradation of cis-naphthalene dihydrodiol (11). The acidic products were not identified in the present study. Absolute configurations of compound III and compound IV. The absolute configuration and ee of compound III formed by JM109(DE3)(pDTG141) were determined by 1H NMR analysis of the diastereomeric boronate esters formed with (2)-(S)and (1)-(R)-MPBA. The methoxyl and methyl groups of the ester formed with (1)-III and (2)-(S)-MPBA had chemical shifts of 3.159 and 1.430 ppm, while the corresponding signals of the (1)-(R)-MPBA ester were observed at 3.238 and 1.305 ppm. The methoxyl signal of the (2)-(S)-MPBA ester of III was shifted upfield (Dd 5 279 ppb) while the methyl signal was shifted downfield (Dd 5 1125 ppb) from the corresponding signals given by the (1)-(R)-MPBA ester. The directional shifts were consistent with the trends in directional shifts previously described (34) and indicated an R configuration for the hydroxyl-bearing benzylic center (C-4) adjacent to the bridgehead carbon position. The 1H NMR spectrum of the ester formed with (2)-(S)-MPBA showed no detectable chemical shift at 3.238 ppm. The results identified compound III as (1)-cis-(3S,4R)-3,4-dihydroxy-3,4,9,10-tetrahydrophenanthrene (.95% ee) (Fig. 2). The absolute configuration of compound IV was determined by correlation of the CD spectra of the compound formed by strains JM109(DE3)(pDTG141) and NCIB 9816-4 with the CD spectra of the individual enantiomers of compound IV reported previously (44) and by conformational analysis. The CD spectra of compound IV formed by both strains expressing NDO exhibited strongly negative absorption at 234 nm and positive absorption at 268 nm (positive Cotton effect) and were identical to the CD spectrum of the (S)-9-hydroxy-9,10-DHP enantiomer reported by Yang and Li (44). The absolute configuration of compound IV was confirmed by determining the conformation of the hydroxyl substituent at position C-9. The pseudoaxial or pseudoequatorial orientation of the C-9 substituent can be related to the skew of the biphenyl chromophore and, in combination with CD spectra, can be used to predict the absolute stereochemistry of 9- or 9,10-substituted 9,10-DHPs [10, 25]. 1H NMR analysis (600 MHz, methanol-d4
APPL. ENVIRON. MICROBIOL.
[10]) of compound IV showed the carbinol proton at 4.81 ppm (doublet of doublets) with J values of 8.11 and 4.83 Hz, indicating a pseudoaxial orientation. Given that the 9-hydroxyl substituent assumes a pseudoequatorial conformation in methanol, the presence of a strong negative CD band at 234 nm (P helicity of the biphenyl chromophore) is indicative of an S configuration for the C-9 position (25) and supports the stereochemical assignment based on the CD spectra previously reported (44). CSP-HPLC analysis showed that compound IV formed by strains NCIB 9816-4 and JM109(DE3)(pDTG141) was enantiomerically pure (.98% ee). The formation of (1)(S)-9-hydroxy-9,10-DHP from DHP (Fig. 2) is consistent with other stereospecific benzylic monohydroxylation reactions catalyzed by NDO which yield secondary alcohols of S configuration (13, 27, 42). The results presented show that NDO catalyzes the enantiospecific cis dihydroxylation of DHA and DHP to yield previously undescribed homochiral cis-diene diols as the major products. The cis-diols formed from both substrates were of R configuration at the benzylic center adjacent to the bridgehead carbon (Fig. 2). The regiochemistry and the absolute stereochemistry of the cis-diols formed from DHA and DHP by NDO are consistent with those formed from naphthalene, anthracene, and phenanthrene (1, 9, 20–22, 24). In contrast to the benzylic oxidation of DHA by strains UV4 (6), PpF39/D, and JM109(pDTG601A) (28), which express TDO, the benzylic 9-monohydroxylation of DHA by NDO was a minor reaction (,5% yield). NDO formed larger amounts of (1)-(S)-9-hydroxy-9,10-DHP (30% yield) by benzylic monooxygenation of DHP (Fig. 2). The formation of (1)-(S)-9-hydroxy-9,10-DHP by wild-type cells of NCIB 9816-4 represents a direct route to this chiral arene hydrate and may provide an alternative to multistep chemical synthesis of this class of compounds from other polycyclic aromatic hydrocarbons (5). The identification of the oxidation products in this study may allow DHA and DHP to serve as diagnostic substrates in the characterization of the oxidation profiles of new and/or hybrid dioxygenases (29). These observations indicate that for DHA and DHP, the location of benzylic carbons influences the predominant reaction(s) catalyzed by NDO. While the type of reaction was not predictable, the absolute configuration of the (9S)-alcohol of DHP and the cis-diols of DHA and DHP were consistent with the stereochemistry of monols and diols formed by NDO from other substrates (13, 22, 24, 27, 32, 41–43). ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grant GM29909 from the National Institute of General Medical Sciences and predoctoral fellowships (to S.M.R.) through the Iowa Center for Biocatalysis and Bioprocessing (University of Iowa) and U.S. Public Health Service Training grant T32 GM8365 from the National Institute of General Medical Sciences. We thank Greg Dekoster for assistance in obtaining CD spectra, John Snyder for acquiring the 600 MHz NMR spectrum of compound IV, and Mahesh Lakshman for helpful discussions. REFERENCES 1. Akhtar, M. N., D. R. Boyd, N. J. Thompson, M. Koreeda, D. T. Gibson, V. Mahadevan, and D. M. Jerina. 1975. Absolute stereochemistry of the dihydroanthracene-cis- and -trans-1,2-diols produced from anthracene by mammals and bacteria. J. Chem. Soc. Perkin Trans. I 1975:2506–2511. 2. Allen, C. C. R., D. R. Boyd, H. Dalton, N. D. Sharma, I. Brannigan, N. A. Kerley, G. N. Sheldrake, and S. C. Taylor. 1995. Enantioselective bacterial biotransformation routes to cis-diol metabolites of monosubstituted benzenes, naphthalene and benzocycloalkenes of either absolute configuration. J. Chem. Soc. Chem. Commun. 1995:117–118. 3. Allen, C. C. R., D. R. Boyd, H. Dalton, N. D. Sharma, S. A. Haughey, R. A. S. McMordie, B. T. McMurray, G. N. Sheldrake, and K. Sproule. 1995. Sul-
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19. 20. 21. 22. 23. 24. 25.
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foxides of high enantiopurity from bacterial dioxygenase-catalyzed oxidation. J. Chem. Soc. Chem. Commun. 1995:119–120. Barnsley, E. A. 1975. The induction of the enzymes of naphthalene metabolism in pseudomonads by salicylate and 2-aminobenzoate. J. Gen. Microbiol. 88:193–196. Boyd, D. R., N. D. Sharma, N. A. Kerley, R. A. S. McMordie, G. N. Sheldrake, P. Williams, and H. Dalton. 1996. Dioxygenase-catalyzed oxidation of dihydronaphthalenes to yield arene hydrate and cis dihydro naphthalenediols. J. Chem. Soc. Perkin Trans. I 1996:67–74. Boyd, D. R., N. D. Sharma, P. J. Stevenson, J. Chima, D. J. Gray, and H. Dalton. 1991. Bacterial oxidation of benzocycloalkenes to yield monol, diol and triol metabolites. Tetrahedron Lett. 32:3887–3890. Brown, S. M., and T. Hudlicky. 1993. The use of arene-cis-diols in synthesis, p. 113–176. In T. Hudlicky (ed.), Organic synthesis: theory and applications. JAI Press, Greenwich, Conn. Carless, H. A. J. 1992. The use of cyclohexa-3,5-diene-1,2-diols in enantiospecific synthesis. Tetrahedron Asymmetry 3:795–826. Catterall, F. A., K. Murray, and P. A. Williams. 1971. The configuration of the 1,2-dihydroxy-1,2-dihydronaphthalene formed in the bacterial metabolism of naphthalene. Biochim. Biophys. Acta 237:361–364. Cobb, D. I., D. A. Lewis, and R. N. Armstrong. 1983. Solvent dependence of the conformation and chiroptical properties of trans-9,10-dihydroxy-9,10dihydrophenanthrene and its monoglucuronides. J. Org. Chem. 48:4139– 4141. Eaton, R. W., and P. J. Chapman. 1992. Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174:7542– 7554. Ensley, B. D., D. T. Gibson, and A. L. Laborde. 1982. Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 149:948–954. Gibson, D. T., S. M. Resnick, K. Lee, J. M. Brand, D. S. Torok, L. P. Wackett, M. J. Schocken, and B. E. Haigler. 1995. Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4. J. Bacteriol. 177:2615–2621. Gibson, D. T., and V. Subramanian. 1984. Microbial degradation of aromatic hydrocarbons, p. 181–251. In D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker Inc., New York. Gibson, D. T., G. J. Zylstra, and S. Chauhan. 1990. Biotransformations catalyzed by toluene dioxygenase from Pseudomonas putida F1, p. 121–132. In S. Silver, A. M. Chakrabarty, B. Iglewski, and S. Kaplan (ed.), Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, D.C. Herbert, A. B., G. N. Sheldrake, P. J. Somers, and J. A. Meredith. 1990. Separation of 1,2-dihydroxycyclohexa-3,5-diene compounds. European Patent EP 0379300A2. Holland, H. L. 1992. Organic synthesis with oxidative enzymes. VCH Publishers, Inc., New York. Hudlicky, T., and J. W. Reed. 1995. An evolutionary perspective of microbial oxidations of aromatic compounds in enantioselective synthesis: history, current status, and perspectives, p. 271–312. In Advances in asymmetric synthesis. JAI Press Inc., Greenwich, Conn. Illman, D. L. 1994. Environmentally benign chemistry aims for processes that don’t pollute. Chem. Eng. News 72:22–27. Jeffrey, A. M., H. J. C. Yeh, D. M. Jerina, T. R. Patel, J. F. Davey, and D. T. Gibson. 1975. Initial reactions in the oxidation of naphthalene by Pseudomonas putida. Biochemistry 14:575–583. Jerina, D. M., J. W. Daly, A. M. Jeffrey, and D. T. Gibson. 1971. cis-1,2Dihydroxy-1,2-dihydronaphthalene: a bacterial metabolite from naphthalene. Arch. Biochem. Biophys. 142:394–396. Jerina, D. M., H. Selander, H. Yagi, M. C. Wells, J. F. Davey, V. Mahadevan, and D. T. Gibson. 1976. Dihydrodiols from anthracene and phenanthrene. J. Am. Chem. Soc. 98:5988–5996. Klecka, G. M., and D. T. Gibson. 1979. Metabolism of dibenzol[1,4]dioxan by a Pseudomonas species. Biochem. J. 180:639–645. Koreeda, M., M. N. Akhtar, D. R. Boyd, J. D. Neill, D. T. Gibson, and D. M. Jerina. 1978. Absolute stereochemistry of cis-1,2-, trans-1,2-, and cis-3,4dihydrodiol metabolites of phenanthrene. J. Org. Chem. 43:1023–1027. Lakshman, M. K., W. Xiao, J. M. Sayer, A. M. Cheh, and D. M. Jerina. 1994.
26. 27.
28. 29.
30. 31. 32.
33. 34.
35.
36.
37.
38. 39. 40. 41. 42. 43.
44.
3359
Synthesis and assignment of absolute configuration to the N6-deoxyadenosine adducts resulting from cis and trans ring-opening of phenanthrene 9,10-oxide. J. Org. Chem. 59:1755–1760. Lee, K., J. M. Brand, and D. T. Gibson. 1995. Stereospecific sulfoxidation by toluene and naphthalene dioxygenases. Biochem. Biophys. Res. Commun. 212:9–15. Lee, K., S. M. Resnick, and D. T. Gibson. 1995. Dioxygenase, monooxygenase, dehydrogenase, and desaturase reactions catalyzed by purified naphthalene dioxygenase, abstr. O-58, p. 378. In Abstracts of the 95th General Meeting of the American Society for Microbiology 1995. American Society for Microbiology, Washington, D.C. Parales, B. P., S. M. Resnick, and D. T. Gibson. Unpublished data. Parales, J. V., R. E. Parales, and S. M. Resnick. 1996. Genetic engineering changes in the substrate oxidation profile of 2-nitrotoluene 2,3-dioxygenase, abstr. Q-329, p. 452. In Abstracts of the 96th General Meeting of the American Society for Microbiology 1996. American Society for Microbiology, Washington, D.C. Rao, S. N., R. A. M. O’Ferrall, S. C. Kelly, D. R. Boyd, and R. Agarwal. 1993. Acid-catalyzed aromatizations of arene oxides and arene hydrates: are arene oxides homoaromatic? J. Am. Chem. Soc. 115:5458–5465. Resnick, S. M., and D. T. Gibson. 1993. Biotransformation of anisole and phenetole by aerobic hydrocarbon-oxidizing bacteria. Biodegradation 4:195– 203. Resnick, S. M., and D. T. Gibson. 1996. Oxidation of 6,7-dihydro-5H-benzocycloheptene by bacterial strains expressing naphthalene dioxygenase, biphenyl dioxygenase, and toluene dioxygenase yields homochiral monol or cis-diol enantiomers as major products. Appl. Environ. Microbiol. 62:1364– 1368. Resnick, S. M., D. S. Torok, and D. T. Gibson. 1993. Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3dioxygenase. FEMS Microbiol. Lett. 113:297–302. Resnick, S. M., D. S. Torok, and D. T. Gibson. 1995. Chemoenzymatic synthesis of chiral boronates for the 1H NMR determination of the absolute configuration and enantiomeric excess of bacterial and synthetic cis-diols. J. Org. Chem. 60:3546–3549. Resnick, S. M., D. S. Torok, K. Lee, J. M. Brand, and D. T. Gibson. 1994. Regiospecific and stereoselective hydroxylation of 1-indanone and 2-indanone by naphthalene dioxygenase and toluene dioxygenase. Appl. Environ. Microbiol. 60:3323–3328. Ribbons, D. W., S. J. C. Taylor, C. T. Evans, S. D. Thomas, J. T. Rossiter, D. A. Widdowson, and D. J. Williams. 1990. Biodegradations yield novel intermediates for chemical synthesis, p. 213–245. In D. Kamely, A. Chakrabarty, and G. S. Omenn (ed.), Biotechnology and biodegradation. Portfolio Publishing Company, The Woodlands, Tex. Sheldrake, G. N. 1992. Biologically derived arene cis-dihydrodiols as synthetic building blocks, p. 127–166. In A. N. Collins, G. N. Sheldrake, and J. Crosby (ed.), Chirality in industry: the commercial manufacture and application of optically active compounds. John Wiley and Sons Ltd., Chichester, United Kingdom. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads; a taxonomic study. J. Gen. Microbiol. 43:159–271. Stinson, S. C. 1995. Chiral drugs. Chem. Eng. News 73:44–74. Suen, W.-C. 1991. Gene expression of naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 in Escherichia coli. Ph.D. dissertation. The University of Iowa City. Torok, D. S., S. M. Resnick, J. M. Brand, D. L. Cruden, and D. T. Gibson. 1995. Desaturation and oxygenation of 1,2-dihydronaphthalene by toluene and naphthalene dioxygenase. J. Bacteriol. 177:5799–5805. Wackett, L. P., L. D. Kwart, and D. T. Gibson. 1988. Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida. Biochemistry 27:1360–1367. Whited, G. M., J. C. Downie, T. Hudlicky, S. P. Fearnley, T. C. Dudding, H. F. Olivo, and D. Parker. 1994. Oxidation of 2-methoxynaphthalene by toluene, naphthalene and biphenyl dioxygenases: structure and absolute stereochemistry of metabolites. Bioorg. Med. Chem. 2:727–734. Yang, S. K., and X.-C. Li. 1984. Direct enantiomeric resolution of cyclic alcohol derivatives of polycyclic aromatic hydrocarbons by chiral stationary phase high-performance liquid chromatography. J. Chromatogr. 291:265– 273.
