APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3323-3328 Vol. 60, No. 90099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Regiospecific and Stereoselective Hydroxylation of 1-Indanoneand 2-Indanone by Naphthalene Dioxygenase

and Toluene DioxygenaseSOL M. RESNICK, DANIEL S. TOROK,t KYOUNG LEE, JOHN M. BRAND,:

AND DAVID T. GIBSON*The Department of Microbiology and Center for Biocatalysis and Bioprocessing,

College of Medicine, The University of Iowa, Iowa City, Iowa 52242

Received 15 April 1994/Accepted 6 July 1994

The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterialstrains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purifiedenzyme components. Pseudomonas sp. strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixtureof 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%o). The (R)-3-hydroxy-1-indanone was formed in62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic. The same cells alsoformed 2-hydroxy-1-indanone from 2-indanone. Purified NDO components oxidized 1-indanone and 2-in-danone to the same products produced by strain 9816/11. P. puta F39/D cells, expressing TDO, oxidized2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone eficiently.Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90%Yo ee (R:S, 5:95) andfailed to oxidize 1-indanone. Oxidation of 1- and 2-indanone in the presence of [18O]oxygen indicated that thehydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation)rather than by the dioxygenation of enol tautomers of the ketone substrates. As alternatives to chemicalsynthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-l-indanoneas the major products from 1-indanone and 2-indanone, respectively.

Toluene dioxygenase (TDO) and naphthalene dioxygenase(NDO) are multicomponent enzyme systems which catalyzethe enantiospecific incorporation of dioxygen into toluene andnaphthalene to form (+)-cis-(lS,2R)-dihydroxy-3-methylcyclo-hexa-3,5-diene (cis-toluene dihydrodiol [9, 15]) and (+)-cis-(lR,2S) -dihydroxy- 1,2-dihydronaphthalene (cis-naphthalenedihydrodiol [14]), respectively. The absolute relationship of thehydroxyl groups is the same in each dihydrodiol, and theconfigurational differences are due to the priorities establishedby the Prelog-Cahn-Ingold sequence rules. Interest in thereactions catalyzed by TDO, NDO, and other bacterial dioxy-genases is related to the use of arene-cis-diols as chiralsynthons in the preparation of a wide range of compounds noteasily obtained by chemical synthesis. Examples include inosi-tols, conduritols, acyclic sugars, and a wide range of biologi-cally active natural products (6, 7, 21).The reactions catalyzed by TDO and NDO are not limited to

cis-hydroxylation of the aromatic nucleus. For example, TDOsfrom Pseudomonas putida Fl and P. putida UV4 catalyze theoxidation of indene (1, 27) and indan (4, 27). In addition, Floxidizes indole (8), and UV4 oxidizes related benzocyclic (4)and heterocyclic (2, 3) aromatic compounds. The substratespecificity of NDO from Pseudomonas sp. strain NCIB 9816has not been examined in detail. However, strain 9816/11, amutant that lacks a functional cis-naphthalene dihydrodioldehydrogenase, oxidizes indan to (S)-1-indanol (96% enantio-meric excess [ee]), whereas P. putida F39/D and UV4, which

* Corresponding author. Phone: (319) 335-7980. Fax: (319) 335-9999.

t Present address: National Institutes of Health, Building 5, RoomBi 31, Bethesda, MD 20892.

t Present address: Department of Biochemistry, University of FortHare, Alice 5700, South Africa.

lack a functional cis-toluene dihydrodiol dehydrogenase, oxi-dize indan to (R)-1-indanol (80 and 98% ee, respectively) (4,27).

Previous reports on the microbial transformation of indanhave made no reference to the formation of hydroxyindanones.Typical microbial transformation products identified fromindan include 1-indanol, 1-indanone, and trans-1,3-indandiol(4, 5, 27). In contrast, it has been reported that when male ratswere treated with indan, the major urinary metabolites formedwere 2-hydroxy-1-indanone and 3-hydroxy-1-indanone in aratio of approximately 2:1 (20, 28).We recently detected the formation of 1-indanone and two

hydroxyindanones as minor metabolites when salicylate-in-duced cells of strain 9816/11 were incubated with (+)-1-indanol for 24 h. This observation, coupled with the fact thatchemical syntheses of 2-hydroxy-1-indanone (17, 26) and 3-hy-droxy-1-indanone (16) require several steps, often with air-sensitive reagents, prompted the present study of the regiospe-cific oxidation of 1- and 2-indanone and the enantiomericcomposition of the hydroxyindanone products formed by NDOand TDO. The results presented provide the first detaileddescription of the bacterial oxidation products formed from 1-and 2-indanone.

MATERIALS AND METHODS

Growth of Pseudomonas strains and transformation of sub-strates. Pseudomonas sp. strain 9816/11 is a spontaneousmutant which oxidizes naphthalene to cis-naphthalene-1,2-dihydrodiol and lacks cis-naphthalene dihydrodiol dehydroge-nase activity (8a). P. putida F39/D is a mutant which oxidizestoluene quantitatively to cis-toluene dihydrodiol (9). Strains9816/11 and F39/D and the wild-type strains NCIB 9816-4 andFl were grown in mineral salts medium (MSB) (22) and

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induced during growth with 0.05% (wt/vol) salicylate andtoluene vapor, respectively. Growth in MSB containing 0.05%(wt/vol) 1- and 2-indanone as the sole carbon source was alsodetermined. Resting cell incubations were carried out usingwashed cells suspended (turbidity, 2.0 to 2.5 at 600 nm) in 50or 500 ml of 50 mM sodium-potassium phosphate buffer (pH7.2), in 250-ml Erlenmeyer or 2.8-liter Fernbach flasks, respec-tively, containing 0.05% of either 1- or 2-indanone and 0.2%(wt/vol) pyruvate. Racemic (synthetic) 2- and 3-hydroxy-1-indanones were provided at 0.01% under identical conditions.After 24 h of incubation (30°C, 220 rpm), cells were removedby centrifugation and the supernatants were extracted threetimes with sodium hydroxide-washed ethyl acetate. Solvent wasdried over anhydrous sodium sulfate (Na2SO4) and concen-trated under reduced pressure (30°C) prior to analysis oftransformation products.

[180]xygen studies with purified NDO and TDO. Theterminal oxygenase ('SPN,) of NDO was purified fromEscherichia coli JM1O9(DE3)(pDTG121) as previously describedby Suen and Gibson (24). ReductaseNAp and ferredoxinNAPwere purified from recombinant E. coli strains expressingindividual or multiple NDO components (23). The ISPTOL andreductaseTOL components of TDO were purified from isopro-pyl-3-D-thiogalactoside (IPTG)-induced cells of E. coli JM109(pDTG601A) which express the cloned todClC2BA genes(29). FerredoxinTOL was purified from JM1O9(pDTG614)(29). Specific details of these purification procedures will bepublished elsewhere. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis analysis showed that all components ofTDO and NDO were homogeneous. When used alone ortogether, the purified reductase and ferredoxin componentsshowed no activity on the substrates used in this study.

Transformation reactions were carried out in 1-dram (3.7-ml) glass vials fitted with Teflon-lined self-sealing septa andscrew-on open-top caps (Altech Associates, Inc., Deerfield, Ill.).The reaction mixtures contained the following in a final volumeof 1.0 ml: 20 ,ug of reductaseNAp, 35 ,ug of ferredoxinNAp, 50 1Lgof ISPNAP, 0.75 mM NADH, and 0.25 mM Fe(NH4)2(SO4)2.6H20 in 50 mM 2-(4-morpholino)ethanesulfonic acid buffer (pH6.8). Concentrations of reductaseTOL, ferredoxinTOL, and 'SPTOLwere 20, 35, and 50 pug, respectively. After flushing theheadspace of each vial with dry nitrogen gas (3 min), 2.0 ml ofthe nitrogen headspace was removed with a gas-tight syringeand replaced with 2.5 ml of 98 atom% ['8O]oxygen (IconIsotopes, Summit, N.J.). The oxygen composition of the head-space of each reaction vial was analyzed at the beginning andend of the experiment. Reactions were initiated by addition of0.5 mM 1-indanone or 2-indanone dissolved in 10 pl ofmethanol. The reaction vials were incubated horizontally at24°C with gentle agitation (60 rpm) for 2 h. The reactions wereterminated by the addition of 2.0 ml of ethyl acetate throughthe Teflon-lined septa. The vials were gently shaken, the ethylacetate was removed, and the aqueous phase was extracted twomore times. The combined extracts were dried over Na2SO4and concentrated (35°C under dry nitrogen) to approximately100 [L prior to analysis (see below).

Analysis, purification, and identification of metabolites.Thin-layer chromatography (TLC) of extracts was performedon silica gel 60 F254 sheets (E. Merck, no. 5735) with chloro-form-acetone (80:20) as the developing solvent. Compoundswere visualized by observing quenching of fluorescence underUV light (254 nm) and by exposure to iodine vapor. Hydroxy-indanones were purified by radial-dispersion chromatography(RDC) using a Chromatotron (Harrison Research, Palo Alto,Calif.). Ethyl acetate extracts were applied to 2.0-mm-thicksilica plates and eluted with a chloroform-acetone step gradi-

ent (0 to 30% acetone; 10% steps over 1 h) at a flow rate of 7ml/min. Fractions (ca. 8 ml) were analyzed by TLC prior tofurther characterization of the different hydroxyindanones.Hydroxyindanones were isolated from enzymic reaction mix-tures by preparative-layer chromatography (PLC) (0.5-mmsilica thickness; Merck) using multiple elution (three develop-ments) with chloroform-acetone (95:5).

N,O-bis-(Trimethylsilyl)trifluoroacetamide with 1% trimeth-ylchlorosilane (BSTFA plus 1% TMSCl; Pierce Chemical,Rockford, Ill.) was used to generate trimethylsilyl (TMS)-etherderivatives. The (S)-(+)- and (R)-(-)-methoxyphenylacetate(O-methylmandelate) esters of 2-hydroxyindanone were syn-thesized essentially as described by Trost et al. (25). Forexample, 2-hydroxy-1-indanone (compound II; 9 mg, 0.061mmol), (S)-(+)- and (R)-(-)-methoxyphenylacetic acids (11mg, 0.067 mmol, 1.1 eq), N,N-dicyclohexylcarbodiimide (13mg, 0.061 mmol, 1.0 eq), and 4-dimethylaminopyridine (1 mg,0.008 mmol, 0.1 eq) were dissolved in 4 ml of methylenechloride, and the mixture was stirred at 22°C for 72 h. Theesters of 3-hydroxy-1-indanone were prepared by treatment ofan anhydrous tetrahydrofuran solution of 3-hydroxy-1-in-danone (compound I; 20 mg, 0.14 mmol) at 0°C under argonwith n-butyl lithium (8 mg, 0.126 mmol, 0.9 eq), and then themethoxyphenylacetyl chlorides (30 mg, 0.16 mmol, 1.2 eq)were added. Reaction mixtures were filtered through Celite545 (Fisher Scientific, Fair Lawn, N.J.) with hexane elution,and the methoxyphenylacetyl esters (Rf value of 0.6 by TLC;chloroform-acetone, 8:2) were isolated by PLC (1.0-mm silica)using chloroform-acetone (95:5) as the developing solvent.Gas chromatography-mass spectrometry (GC-MS) was per-

formed on a Hewlett-Packard model 5890 gas chromatographequipped with a Hewlett-Packard Ultra-1 capillary column(inside diameter, 0.2 mm by 25 m; film thickness, 0.33 pLm).The column temperature was programmed from 70 to 240°C at10°C/min with a helium flow of 25 cm/s. Temperatures of theinjection port and transfer line were 220 and 280°C, respec-tively. Samples (1 [L) were injected at a split ratio of 50:1, andmass spectra were obtained using a Hewlett-Packard model5970 mass selective detector with electron impact ionization(70 eV). Headspace atmosphere (for [180]oxygen incorpora-tion studies) was similarly analyzed, except that the columntemperature was 150°C.

Chiral stationary-phase high-performance liquid chromatog-raphy (CSP-HPLC) was performed with a Waters Associates510 solvent pump, a U-6K injector, a model 490E multiwave-length detector, and Maxima 820 workstation software (foracquisition of data and peak integration). Enantiomers wereseparated on a Chiralcel OB column (25 by 4.6 cm; ChiralTechnologies, Exton, Pa.) using a mobile phase of hexane and2-propanol (9:1) at a flow rate of 0.5 ml/min. The detectormonitored wavelengths at 210, 254, 270, and 280 nm. Underthese conditions, the (R)- and (S)-enantiomers of 3-hydroxy-1-indanone eluted with retention volumes (R,) of 11.9 and14.3 ml, respectively. The (R)- and (S)-2-hydroxy-1-indanoneenantiomers eluted in the same order, at 12.6 and 17.1 ml,respectively.

Proton ('H) and carbon ("3C) nuclear magnetic resonance(NMR) spectra were recorded on a Bruker WM-360 spectrom-eter at 360.14 and 90.56 MHz, respectively. Chemical shifts (8)are reported in parts per million with respect to tetramethyl-silane, and coupling constants (J values) are given in hertz.Abbreviations for 'H NMR signals are as follows: s, singlet; d,doublet; t, triplet; dd, doublet of doublets; and m, multiplet.Optical rotations at 25°C were determined using a Perkin-Elmer model 141 polarimeter. Absorption spectra were re-

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HOMe

H

H Hb

- alcoho 0

S alcohol

HOMe

Ha

H

0

0 If 0

H*

R alcohol

(R) H.

(S) Hb (S) H,

3.30 3.20 3. 10 3.00 2.90 2.60 2. 70 2.60 2.50 2. 40

FIG. 1. Portion of the 'H NMR spectrum (CDCl3) of the (R)-(-)-methoxyphenylacetate ester of 3-hydroxy-1-indanone formed by strain9816/11. The signals corresponding to the diastereomeric esters of (R)-3-hydroxy-1-indanone (dd, 2.65 and 3.20 ppm) and (S)-3-hydroxy-1-indanone (dd. 2.45 and 3.08 ppm) are indicated. Extended Newman projections have the intervening ester linkage omitted to make a more

convenient view of the esters. See text for discussion of the method and results.

corded over the 200- to 350-nm range using a Beckman DU-70spectrophotometer.

Syntheses of 3- and 2-hydroxy-1-indanone. (±)-3-Hydroxy-1-indanone was prepared from 1,3-indandione in glacial aceticacid by treatment with zinc dust as described by Lacy et al.(16). (+)-2-Hydroxy-1-indanone was synthesized by treatingthe TMS-enol ether of 1-indanone with m-chloroperoxyben-zoic acid (19). The enol ether was prepared as previouslydescribed (13) by treatment of a tetrahydrofuran solution of1-indanone at -78°C under argon with lithium diisopropylam-ide and then by quenching with TMSCl-triethylamine.

RESULTS

Growth studies and transformations by wild-type and mu-

tant strains. Pseudomonas sp. strains 9816-4, 9816/11, Fl, andF39/D grew well on 2-indanone but not on 1-indanone (datanot shown). Transformation of 1- and 2-indanone by inducedand uninduced cells of strains 9816-4 and Fl gave the same

hydroxyindanone products observed with strains 9816/11 andF39/D; however, various additional products were detected.For this reason, mutant strains 9816/11 and F39/D, as well as

purified NDO and TDO components, were used to focus onthe initial hydroxylation reactions.

Transformations catalyzed by Pseudomonas sp. strain 9816/11. Incubation of salicylate-induced cells (500 ml) of Pseudo-monas sp. strain 9816/11 with 1-indanone led to the accumu-lation of two polar products which showed Rf values of 0.4(compound I) and 0.5 (compound II) by TLC in chloroform-acetone (8:2). GC and 'H NMR analysis showed that com-

pounds I and II were formed in a ratio of approximately 9:1.Compound I (52 mg) was isolated by RDC and had thefollowing characteristics: GC-MS, retention time (R,) of 12.8min, molecular ion [M+ (% relative intensity)] at m/z 148 (100)with major fragment ions at m/z 131 (15), 119 (37), 105 (53),

103 (24), 91 (46), 77 (46), 65 (13), 51 (35), and 50 (32); 'HNMR (CDCl3) 8 2.6 (dd, J = 18.9, 2.4 Hz, 1H), 3.11 (dd, J =

18.9, 6.8 Hz, 1H), 5.41 (dd, J = 6.5, 2.3 Hz, 1H), 7.48 (t, J = 6.9Hz, l H), 7.65-7.73 (m, 3H); '3C NMR (CDCl3) 8 46.96 (CH2),67.86 (CH), 122.5 (CH), 126.4 (CH), 129.0 (CH), 135.0 (CH),136.6 (C), 156.8 (C), 203.0 (C=O); UV spectrum (MeOH)Xmax, 206, 243, 280 nm; [oL]D-87.5' (c 2.0, CHC13). These data(with the exception of optical rotation) are identical to those ofsynthetic ( + )-3-hydroxy-1-indanone, and our mass spectraldata are in agreement with those previously reported (28).During GC-MS analysis, up to 16% of the total ion currentarea consisted of a compound with an R, of 8.7 min and an M+at m/z 130. This compound was identified as 1-indenone andwas shown to result from dehydration of compound I in the GCinlet. When the injection port was lowered to 140°C (from220°C), less than 1% 1-indenone was produced.The absolute configuration and enantiomeric composition of

compound I formed by strain 9816/11 were determined by 'HNMR analysis of its (R)- and (S)-methoxyphenylacetyl esters(25). Trost et al. have shown that the methoxyphenylacetylesters of secondary alcohols assume a conformation in whichthe oxygen atoms are in alignment (see Fig. 1), most likelybecause of electronic effects (25). When this occurs, the phenylgroup will eclipse substituents on either side of the alcohol,depending on the stereochemistry of the alcohol. The substitu-ent which is eclipsed by the phenyl ring is then shifted upfieldas a result of the shielding it experiences by the phenyl ring.The 'H NMR spectrum (CDCl3) of the ester formed with

(R)-methoxyphenylacetic acid (Fig. 1) revealed the presence oftwo sets of signals corresponding to the diastereomeric estersof (R)-3-hydroxy-1-indanone (8 2.65 [dd, J = 19.0, 3.0 Hz, 1H],3.20 [dd, J = 19.0, 7.3 Hz, 1H], 3.38 [s, 3H], 4.75 [s, 1H], 6.26[dd,J = 7.0, 2.9 Hz, 1H], 7.07-7.65 [m, 9H]) and (S)-3-hydroxy-1-indanone (8 2.45 [dd, J = 19.0, 3.0 Hz, 1H], 3.08 [dd, J =

19.0, 7.3 Hz, 1H], 3.35 [s, 1H], 4.72 [s, 3H], 6.30 [dd,J = 7.0, 2.9

° / dMeo

lee

(R) Hb

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TABLE 1. Products formed from 1-indanone and 2-indanone by induced whole cells of strains 9816/11 and F39/D

Strain Substrate Product Relative yield (%)' Enantiomeric composition (%)*

9816/11 1-Indanone 3-Hydroxy-1-indanone 91 81 (R)-3-hydroxy-1-indanone2-Hydroxy-1-indanone 9 51 (R)-2-hydroxy-1-indanone

9816/11 2-Indanone 2-Hydroxy-1-indanone 99 53 (S)-2-hydroxy-1-indanone2-Hydroxy-2-inden-1-one 1 NAC

F39/D 2-Indanone 2-Hydroxy-1-indanone 72-96 88 (S)-2-hydroxy-1-indanone2-Hydroxy-2-inden-1-one 4-28 NA

a Relative product yields were determined from peak area integrations of total ion current chromatograms.b The enantiomeric compositions of isolated hydroxyindanones were determined by integrations of CSP-HPLC peak areas of the individual enantiomers. The

individual enantiomers were collected and shown to have superposable UV spectra.c NA, not applicable.

Hz, 1H], 7.07-7.65 [m, 9H]). The downshifted signals (dd at2.65 and 3.20 ppm; Fig. 1) for the (R)-methoxyphenylacetylester indicated that the hydroxyl group at carbon position C-3was predominantly of the (R)-configuration. For the (S)-methoxyphenylacetyl ester, the magnitude of the integrals ofthe same signals were reversed relative to those of the (R)-ester (data not shown). The enantiomeric composition of theproduct, determined by integration of the proton signalsarising from the two diastereomers, was 80% (R)-3-hydroxy-1-indanone. CSP-HPLC was also used to determine the enan-tiomeric purity of compound I. On the basis of the integrationof peak areas, the enantiomeric composition was shown to be81% (R)-3-hydroxy-1-indanone (62% ee) (Table 1).Compound II (5 mg) was a minor product formed from

1-indanone by strain 9816/11. The product was isolated by PLCand characterized by GC-MS, 'H and 13C NMR, and UVspectral analyses: GC-MS, R, of 11.6 min and M+ (% relativeintensity) at m/z 148 (100) with major fragment ions at m/z 131(14), 119 (60), 105 (29), 91 (87), 77 (16), 65 (34), 51 (21), and50 (24); 'H NMR (CDCl3), 8 3.03 (dd, J = 16.5, 5.1 Hz, 1H),3.58 (dd, J = 16.5, 7.9 Hz, 1H), 4.54 (dd, J = 7.9, 5.1 Hz, 1H),7.42 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.64 (t, J =7.5 Hz, 1H), 7.75 (d,J = 7.6 Hz, 1H); 13C NMR (CDCl3) 8 35.1(CH2), 74.1 (CH), 124.3 (CH), 126.7 (CH), 127.9 (CH), 134.0(C), 135.8 (CH), 150.9 (C), 206.8 (C=O); UV spectrum(MeOH) km., 207, 246, and 292 nm. The above data wereidentical with those of synthetic 2-hydroxy-1-indanone. Incontrast to compound I, compound II did not dehydrate underthe GC conditions used. CSP-HPLC separated the enanti-omers of compound II and showed that compound II formedby strain 9816/11 from 1-indanone was essentially racemic(51:49; Table 1).When salicylate-induced cells (500 ml) of strain 9816/11

were incubated with 2-indanone, a single product was observedby TLC, with an Rf value of 0.5. The product (65 mg) wasisolated by RDC, and analysis by GC-MS, 'H NMR, and 13CNMR showed that this compound was identical to compoundII. CSP-HPLC resolved two enantiomers and showed that(S)-2-hydroxy-1-indanone was in slight excess (6% ee) (Table1). GC-MS analysis of ethyl acetate extracts also detected aminor product (compound III) in trace amounts (Table 1).This compound was identified in reactions with F39/D (seebelow).

Salicylate-induced cells of strain 9816/11 were also incubatedwith synthetic (±)-3-hydroxy-1-indanone and (±)-2-hydroxy-1-indanone to determine if a dehydrogenase activity influencedthe enantiomeric compositions of these products. After 24 h ofincubation, CSP-HPLC analysis showed that the compositionof the 3-hydroxy-1-indanone was still racemic while (S)-2-hydroxy-1-indanone was found in slight excess (54:46) (datanot shown).

Transformations catalyzed by P. putida F39/D. Toluene-induced cells (50 ml) of strain F39/D showed extremely poortransformation of 1-indanone to compound I (4% yield byGC-MS at 24 h). However, the same cells (500 ml) catalyzedefficient conversion of 2-indanone to a product identified(GC-MS, 'H NMR, and 13C NMR) as compound 11 (72 to 96%yield; Table 1). Compound II (73 mg, [OLID +21° [c 1.4,CHCl3]) was isolated by RDC and derivatized with bothmethoxyphenylacetyl chlorides. 'H NMR analysis of (R)-(-)-and (S)-(+)-methoxyphenylacetyl esters of compound IIshowed two sets of signals corresponding to the diastereomericesters of (R)-2-hydroxy-1-indanone (CDCl3 8 3.05 [dd, J =16.9, 4.4 Hz, 1H], 3.50-3.78 [m, 2H], 4.91 [s, 3H], 5.47 [dd, J =8.1, 4.9 Hz, 1H], 7.28-7.81 [m, 9H]) and (S)-2-hydroxy-1-indanone (CDCl3 8 2.77 [dd, J = 16.9, 4.4 Hz, 1H], 3.50-3.78[m, 2H], 4.93 [s, 3H], 5.53 [dd, J = 8.1, 4.9 Hz, 1H], 7.28-7.81[m, 9H]). Integration of the signals (dd at 2.77 and 3.05 ppm)indicated that (S)-2-hydroxy-1-indanone was formed in ee byF39/D (data not shown). CSP-HPLC analysis confirmed theenantiomeric composition of compound II as 88% (S)-2-hydroxy-1-indanone (76% ee; Table 1). In a separate experi-ment, toluene-induced cells of PpF39/D were incubated for 24h with synthetic (±)-2-hydroxy-1-indanone. CSP-HPLC anal-ysis of the recovered substrate showed its composition to be(S)-2-hydroxy-1-indanone of 54% ee (data not shown).A minor product (compound III) formed from 2-indanone

by both F39/D and 9816/11 cells was detected by GC-MS (R, of12.6 min) and showed a M+ (% relative intensity) at m/z 146(13) with fragment ions at m/z 118 (74), 90 (100), 89 (55), 74(4), 63 (34), 51 (11), 39 (15), and 27 (6). Compound III formeda TMS derivative which showed a M+ (% relative intensity) atm/z 218 (19) and fragment ions at m/z 205 (40), 147 (61), 133(20), 117 (33), 103 (27), 73 (100), 59 (12), and 45 (22). Theformation of a TMS derivative indicated the presence of anexchangeable hydroxyl proton and supports the identificationof compound III as 2-hydroxy-2-inden-1-one.

Oxidation of 1- and 2-indanone by purified NDO and TDOcomponents in the presence of [180]oxygen. 1-Indanone and2-indanone were incubated with purified NDO and TDOcomponents in the presence of an atmosphere containing.90% ['8O]oxygen. Product distribution was determined byGC-MS of reaction extracts. Enantiomeric compositions of thePLC-isolated products were determined by CSP-HPLC. Thereaction products formed and their normalized '8O-labeledisotopic enrichment are summarized in Table 2. Mass spectraof all hydroxyindanones formed in the presence of [t8O]oxygenshowed M+ + 2 at m!z 150, indicating the incorporation of asingle atom of molecular dioxygen. The lack of any M+ + 4 ionindicated that the possibility of product formation throughdioxygenation of the enol tautomer of either substrate wasunlikely. Purified NDO oxidized 1-indanone to 78% (R)-3-

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TABLE 2. Incorporation of ['80]oxygen into 1-indanone and 2-indanone by purified NDO and TDO components andenantiomeric purity of the products

Product Relative Isotopic ['80]oxygen Product [180]oxygen NormalizedEnzyme Substrate (enantiomeric composition [%])" yield (%) enrichment (%) enrichment (%)b enrichment (%)'

NDO I-Indanone (R)-3-Hydroxy-l-indanone (78) 87 90 92 100(R)-2-Hydroxy-l-indanone (61) 13 90 88 98

NDO 2-Indanone (S)-2-Hydroxy-l-indanone (88) 100 92 77 84TDO 2-Indanone (S)-2-Hydroxy-l-indanone (95) 100 91 60 66

" Product identification based on GC-MS characteristics with relative product yields determined from peak area integrations of total ion current chromatograms. Noproducts were detected in 2-h reaction mixtures containing 1-indanone and TDO components. Enantiomeric composition was determined from integrations ofCSP-HPLC resolved peaks corresponding to the enantiomers of the isolated products.

b Determined by the intensities of the m/z at (M+ + 2)/[M+ + 2)] x 100.c Defined as (['80]oxygen enrichment of product/['80]oxygen enrichment of headspace) x 100.

hydroxy-1-indanone (87% yield, 56% ee) and 61% (R)-2-hydroxy-1-indanone (13% yield, 22% ee). Purified TDO didnot oxidize 1-indanone. Both TDO and NDO oxidized 2-in-danone to (S)-2-hydroxy-1-indanone (100% yield); however,the enantiomeric purity of the product formed by TDO (90%ee) was higher than that from NDO (76% ee) (Table 2).2-Hydroxy-2-inden-1-one (compound III) was not formed bypurified NDO and TDO components.

DISCUSSIONTDO and NDO have relaxed substrate specificities and are

capable of catalyzing dioxygenation and monooxygenationreactions with different substrates. Thus, TDO oxidizes indanto (R)-1-indanol, while NDO forms the (S)-enantiomer (27).NDO seems to be more versatile than TDO, since it has theability to catalyze the desaturation of indan to indene (12) andphenetole to ethenyloxybenzene (18). In addition, NDO cata-lyzes O-dealkylation reactions with anisole and phenetole,while TDO does not show desaturase or O-dealkylation activitywith phenetole (18).The present study provides further examples of monooxy-

genation reactions catalyzed by NDO and TDO. Salicylate-induced cells of strain 9816/11 oxidized 1-indanone to (R)-3-hydroxy-1-indanone and 2-hydroxy-1-indanone as the majorand minor products, respectively (Table 1). Purified NDOformed the same products (Table 2) (Fig. 2A). In contrast,toluene-induced cells of F39/D and purified TDO did notoxidize 1-indanone efficiently. This illustrates further differ-ences in the substrate specificities of NDO and TDO.

Induced cells of strain 9816/11 and F39/D oxidized 2-in-danone almost exclusively to (S)-2-hydroxy-1-indanone. Aminor product formed by both organisms was identified as2-hydroxy-2-inden-1-one (Table 1). The latter product mayresult from dehydrogenation or desaturation of the 2-hydroxy-1-indanone. However, on the basis of the observed product, itis difficult to determine whether desaturation or dehydrogena-tion is occurring, since 1,2-indandione and 2-hydroxy-2-inden-1-one are merely keto-enol tautomers and thus offer littleinsight into the mechanism of the oxidation. Purified NDO andTDO both oxidized 2-indanone to (S)-2-hydroxy-1-indanone asthe only detectable product (Table 2) (Fig. 2B).The 3-hydroxy-1-indanone formed by strain 9816/11 and

NDO was the (R)-enantiomer (ca. 60% ee). In contrast,induced cells of strain 9816/11 and purified NDO showed lessenantiospecificity in the formation of (R)-2-hydroxy-1-in-danone (Tables 1 and 2).The 2-hydroxy-1-indanone formed from 2-indanone by in-

duced cells of strains 9816/11 and F39/D was the (S)-enanti-omer, with F39/D showing a higher enantiospecificity thanstrain 9816/11 (Table 1). The same pattern was observed with

purified NDO and TDO, although NDO produced (S)-2-hydroxy-1-indanone in 76% ee compared with the 6% ee forinduced cells of strain 9816/11. The reason for this differencewas not pursued further in the present study.

Experiments with [18O]oxygen showed that for 1-indanone,the single atom of oxygen incorporated into (R)-3-hydroxy-1-indanone and (R)-2-hydroxy-1-indanone by purified NDO wasderived exclusively from dioxygen (Table 2). This suggests thatthe hydroxylation reaction is tightly coupled. In contrast, asignificant percentage of the oxygen in the (S)-2-hydroxy-1-indanone formed from 2-indanone by NDO and TDO was notfrom molecular oxygen and was presumed to be derived fromwater. This suggests that an iron-bound oxygen species in theoxygenase components of NDO and TDO may be the site ofexchange of oxygen with water. A similar exchange reactionmay also be involved in the oxidation of indan to (R)-1-indanolby TDO (27).The formation of 3-hydroxy-1-indanone and 2-hydroxy-1-

indanone as the major bacterial oxidation products from 1- and2-indanone, respectively, offers a biocatalytic alternative tomultistep chemical syntheses of these compounds. In addition,the characterization of these oxidation products may aid inidentification of metabolites in the bacterial catabolic pathwaysof larger indeno-aromatic hydrocarbons. In fact, an unidenti-fied metabolite (compound IV) with an M+ at m/z 148 formedfrom fluorene by anArthrobacter species (strain F101) (10) has

A

0

1-Indanone

0

NDO

H OH

(R)-3-Hydroxy-l-indanone

0

H

H

II(R)-2-Hydroxy-l-indanone

B

OO0=o

2-Indanone

0

NDO & TDO cI iC

II(S)-2-Hydroxy-l-indanone

FIG. 2. (A) Products formed from 1-indanone by NDO; (B) oxi-dation of 2-indanone to (S)-2-hydroxy-1-indanone by NDO and TDO.The enantiomeric compositions of compounds I and II are given inTable 2.

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3328 RESNICK ET AL.

spectral data (1H NMR, MS, and UV data) which are essen-tially identical to those reported for 3-hydroxy-1-indanone inthe present study. Also, 1-indanone was recently identified as ametabolite in the degradation of fluorene by Pseudomonascepacia F297 (11). Hence, the 3-hydroxy-1-indanone formed bystrain F101 could conceivably result via hydroxylation of1-indanone in the manner demonstrated for NDO.

ACKNOWLEDGMENTS

We thank D. L. Galinis for bringing the methodology of Trost et al.to our attention and for useful discussions about stereochemicaldeterminations.

This work was supported in part by U.S. Public Health Service grantGM29909 from the National Institute of General Medical Sciences.S.M.R. is a predoctoral fellow supported by U.S. Public Health ServiceTraining grant T32 GM8365 from the National Institute of GeneralMedical Sciences, and J.M.B. was supported by a grant from the DowChemical Co. (Midland, Mich.).

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