THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, · PDF fileis the dominant P450 reaction...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 268, No. 14, Issue of May 15, pp. 9986-999’7,1993

Printed in U. S. A .

Partitioning between N-Dealkylation and N-Oxygenation in the Oxidation of N,N-Dialkylarylamines Catalyzed by Cytochrome P450 2B1*

(Received for publication, August 17, 1992, and in revised form, February 4, 1993)

Yasuo Setot and F. Peter Guengerichs From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Mediciw, Nashville, Tennessee 37232

Aminium radicals have been proposed as intermediates in amine N-dealkylations and N-oxygenations catalyzed by cytochrome P450 (P450) and some other enzymes. P450s can form some N-oxides and hydroxylamines but these are not favored whenever N-dealkylation is possible. However, if a paradigm involving l-electron oxidation is valid, then some finite level of partitioning of aminium radicals between N-oxygenation and N-dealkylation might be expected in all cases. Methods were developed for the selective and sensitive measurement of N,N-dialkylaniline N-oxides using high performance liquid chromatography, radiochro- matography, and TiC13 reduction. These N-oxides were relatively stable in the presence of P450 2B1. In the presence of NADPH and NADPH-P45O reductase some reduction to N,N-dialkylamines occurred, along with N-dealkylation (to monoalkylanilines); there was also slow N-dealkylation in the absence of NADPH, which is interpreted in terms of homolytic scission of the N - 0 bond; N,N-dialkylanilines were not formed nor did the N-oxides support other oxygenation reactions. P450 2B1 (with its reductase and NADPH) formed N-oxides at low rates from several N,N-dialkylaniline derivatives, including N,N-dimethylaniline, N,N-diethylaniline, N-ethyl-N-methylaniline, 4- methyl-N,N-dimethylaniline, 4-cyano-N,N-dimethylaniline, N-phenylpyrrolidine, and N,N-dimethyl-2- aminofluorene. The ratio of N-dealky1ation:N-oxygenation varied from 1020 to 6 in this series. These results are consistent with the view that aminium radicals are a branch point in N-oxygenation and N-dealkylation reactions catalyzed by metalloproteins, although some alternate explanations cannot be ruled out. While N- dealkylation is the dominant process in all of the P450- catalyzed amine oxidations, there should be a finite partition ratio between these reactions depending upon the particular enzyme and substrate. The N-oxygenation reaction is probably more complex than a direct radical recombination event and is postulated to involve one or more intermediates.

*This research was supported in part by United States Public Health Service Grants CA 44353 and ES 00267. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported in part by a fellowship from the Science and Tech- nology Agency of the Government of Japan. Current address: 2nd Chemistry Section, National Research Institute of Police Science, 6 Sanban-cho, Chiyoda-ku, Tokyo 102, Japan.

To whom correspondence should be addressed Dept. of Biochem- istry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141.

Amine oxidations are important in the metabolism of both chemicals endogenous to cells and those introduced as drugs, pesticides, etc. (Walsh, 1979). In eukaryotic systems many of these oxidations are localized in the endoplasmic reticulum and there has been considerable interest in the oxidations catalyzed by the hemoprotein P4501 (Guengerich, 1990) and the flavoprotein termed microsomal flavin-containing monooxygenase (Ziegler, 1991). Both of these enzymes are actually families of related proteins, but there is considerable evidence to support the view that the basic catalytic mechanisms are rather general within each group. The prevailing view is that the P450s generally catalyze N-dealkylation of amines when possible and that the microsomal flavin-containing monooxygenases catalyze N-oxygenation (Ziegler, 1991; Guengerich, 1990).

An early view was that N-oxides might be intermediates in the overall N-dealkylation of alkylamines (Ziegler and Pettit, 1964). However, most N-oxides are relatively stable (e.g. sparteine N-oxides (Guengerich, 1984)) and would not be catalytically competent as intermediates (with some exceptions such as in the Cope elimination (Cashman, 1989)). An alternate and most likely intermediate in the mechanism is a carbinolamine, first suggested by Brodie et al. (1958). In recent years the concept has been advanced that a key step in N- oxidation is the formation of an aminium radical (Guengerich, 1990; Burka et al., 1985; Macdonald et al., 1982,1989; August0 et al., 1982; Guengerich et al., 1984; Bondon et al., 1989; Miwa et al., 1983; Guengerich and Bocker, 1988) (Scheme l), which gives the carbinolamine after oxygen rebound. N-Dealkyla- tion can result from a-deprotonation of the aminium radical (Scheme 1, step indicated by kd) , while N-oxygenation could result from radical recombination (step indicated by k,,). The situation with the microsomal flavin-containing monooxygenase differs in that the key intermediate is considered to be the flavin 4a-hydroperoxide, which is prone to a direct heterolytic attack by the amine to form the N-oxide in a con- certed reaction (Ziegler, 1991).

Previous work has advanced the view that N-dealkylation is the dominant P450 reaction observed with N-alkylamines because of the presumed high acidity of a-hydrogens of aminium radicals (Guengerich and Macdonald, 1984). N-Oxygen- ation was considered to be expected in only three cases: (i) no a-hydrogens are present (e.g. arylamines (Frederick et al., 1982)); (ii) a-hydrogens are present but cannot be abstracted because of Bredt’s rule (e.g. quinidine (Guengerich et al.,

The abbreviations used are: P450, cytochrome P450 (also termed heme-thiolate protein P450 (Palmer and Reedijk, 1992)); HPLC, high performance liquid chromatography; TLC, thin layer chromatography; FAB, fast atom bombardment; FAB+-MS, (positive ion) fast atom bombardment-mass spectometry.

9986

SCHEME 1. Postulated events in P46O-catalyzed amine oxidation.

N-Oxide Formation and Decomposition by P450

0

(bo)" RR'NCH2R" - k. (bo)'* RR'NCH2R"& .+ RR'NCH,R" 4 -1 e-

9987

1986)); or (iii) a-hydrogens are present but the presence of a neighboring electron donor stabilizes the putative aminium radical (e.g. azoprocarbazine (Prough e t al., 1984)). However, in more recent years there have been at least three important exceptions to this view. Methamphetamine forms the hydroxylamine and then a nitrone (Baba et al., 1987); the pyrrolizi- dine alkaloid senecionine is converted to an N-oxide (Wil- liams et d . , 1989); N-(1-phenylcyclobuty1)benzylamine is oxidized to the hydroxylamine and then to the nitrone (Bondon et al., 1989). These reactions may all be considered in terms of the competition of pathways emanating from the aminium radical in Scheme 1. Other important developments relevant to the mechanism of P450-catalyzed N-oxygenation need to be considered. Experiments with carbon "radical clocks" in several laboratories have indicated that rates of radical recombination (oxygen rebound) are quite rapid, on the order of lo9 s" (Ortiz de Montellano and Stearns, 1987; Bowry and Ingold, 1991). Thus we might expect these rates to be applicable to the Fe(1V) = O/aminium radical pairs. Theoretical (Nelsen and Ippoliti, 1986) and experimental (Dinnocenzo and Ban- ach, 1989; Okazaki and Guengerich, 1993) studies have been presented to argue that the a-hydrogens of aminium radicals are not as acidic as previously thought. With these consider- ations in mind, one might expect to observe a finite level of N-oxygenation in all P450-catalyzed amine oxidations if the postulated Scheme 1 is a true paradigm for these reactions.

We selected N,N-dimethylaniline as the basic structure to be used in these studies, since it provided a case where extensive N-demethylation occurs and the literature regarding the production of N-oxides is not clear. Hlavica and Hulsmann (1979) reported that rabbit P450 1A2 was able to convert this substrate to the N-oxide with a kcat of 1-2 min-', although the rate decreased with further purification of the NADPH-P450 reductase component and controls regarding H202 involvement were lacking. On the other hand, Pandey et al. (1989) did not detect formation of the N-oxide at a rate of >0.3 min" with rabbit P450 1A2 or any of several other P450s. Our studies on N,N-dimethylaniline were extended to a group of compounds having this basic structure contained within them.

In the course of measurement of levels of N-oxides we necessarily considered the fates of these compounds. We had previously examined N,N-dimethylaniline N-oxides and found that these were relatively stable in the presence of P450 (Burka et al., 1985). However, a slow but finite rate of N- dealkylation of N,N-dimethylaniline N-oxides does occur. We had interpreted this in terms of a mechanism in which the iron is oxidized to the formal Fe(1V) state instead of Fe(V) (Fe03+). However, others have argued for heterolytic (as opposed to homolytic) cleavage of the N-0 bond to yield the formal Fe(V) state, primarily on the basis of biomimetic models (Heimbrook e t al., 1984; Ostovic e t al., 1987). This matter was reconsidered, and the kinetics of several reactions are interpreted in favor of the homolytic cleavage mechanism (Scheme 2).

Evidence is presented that the general paradigm involving aminium radicals (Scheme 1) is appropriate for both P450-

PH (F.oH)~* R R ' N ~ H R " ~ RR'NCHW- RR'NH + OHCR"

catalyzed N-dealkylation and N-oxygenation. There is a finite partition ratio and in simple alkylamines such as N,N-dialkylaniline derivatives, some N-oxide is formed even in the presence of accessible a-protons. However, the N-oxygenation process is probably more complex than a direct transfer of oxygen to the aminium radical.

EXPERIMENTAL PROCEDURES

Chemicals

Aniline, N-methylaniline, N-ethylaniline, N,N-dimethylaniline, N-ethyl-N-methylaniline, N,N-diethylaniline, N,N-dimethyl-p-toluidine, p-aminobenzonitrile, p-(N,N-dimethylamino)benzonitrile, 2- aminofluorene, N,N-dimethyl-2-aminofluorene, m-chloroperbenzoic acid, CH31, (C,H&N, and TiCl, were obtained from Aldrich. N - Methyl-p-toluidine, (CzH&NH, and Chromagram sheet 13181 (silica gel adsorbent with fluorescent indicator) were obtained from Eastman Kodak Co. (Rochester, NY). N-Phenylpyrrolidine was purchased from Lancaster/MTM Research Chemicals (Windham, NH). L-W 1,2-Dilauroyl-sn-glycero-3-phosphocholine, HZOZ (30% solution), catalase (from bovine liver), superoxide dismutase (from bovine eryth- rocytes), L-glutathione, and N,N-dimethyl[ring-'4C]aniline (specific activity 1.9-6.2 mCi mmol") were obtained from Sigma. Alumina (basic, Brockman activity I) and Scinti-Verse I1 liquid scintillation mixture were obtained from Fisher Scientific Co. Silicic acid (60-200 mesh), silica gel TLC plates with fluorescent indicator, and p-toluidine were obtained from J. T. Baker Inc. (Phillipsberg, NJ). Sep-Pak C18 (octadecylsilane) cartridges were from Waters Associates Inc. (Milford, MA). All other chemicals were analytical reagent grade.

Commercial N,N-dialkylanilines were purified by applying material (5 gin n-hexane) to a silicic acid column (2.2 X 25 cm) equilibrated with n-hexane and eluting (N,N-dialkylanilines) with n-hexane; n- hexane was evaporated in uacuo. Commercial N,N-dimethyl[ring-'4C] aniline was dissolved in CH30H (to 100 pCi m1-I) and applied to a preparative HPLC column (Ultremex 5 C18 octadecylsilane, 5 pm, 10 x 250 mm; Phenomenex, Torrance, CA), using an elution solvent consisting of CH,OH/H,O (8020, v/v) and a flow rate of 4.0 ml min". The N,N-dimethylaniline fraction ( t ~ 6-7 min) was collected and extracted into CH,C1, (at alkaline pH). The organic phase was dried with Na2S04 and evaporated to dryness under N2 after the addition of H3P04 to acidic pH. The resulting dried sample was dissolved in Hz0 and used as substrate. All amines and amine oxides used in incubations were judged to be >99.7% pure by HPLC prior to use. Radiolabeled substrate was >99.9% pure.

Syntheses

4-(N-Methylarnino)benzonitri~e-CH31(3.4 g, 24 mmol, 1.5 ml) was added to a (CH&CO solution of p-aminobenzonitrile (0.59 g, 5.0 mmol) and NaHC0, (1.0 g, 12 mmol) and stirred while heating under reflux for 30 h. Analytical TLC of the reaction mixture indicated a mixture of the primary amine and the N-methylaniline and N,N- dimethylaniline derivatives. The reaction mixture was filtered, and the resulting yellow (CH&CO solution was evaporated to dryness in

bexane/CHCl~/(C,H,),NH, 5:4:1, v/v/v; Baker). The middle of the uacuo. The sample was purified by TLC using silica gel plates (n-

three zones detected under UV light ( R R 0.42) was collected and 4- (N-methy1amino)benzonitrile was eluted with CH30H. The resulting material was purified by HPLC (Ultremex 5 C18,5 pm, 10 X 250 mm) using a mixture of CH,OH/H,O (60:40, v/v) and a flow rate of 4.0 ml min". The fraction of interest ( t R 4.0-6.5 min) was collected and extracted into CHzClz at alkaline pH. The organic phase was dried with NazS04 and evaporated to give crystalline 4-(N-methylam- in0)benzonitrile: m.p. 89-89.5 "C; 'H NMR (C2HCl3) d 2.88 ppm (s, ,H, N-CH,), 4.24 (sb, lH, NH), 6.55 (d, 2H, o-phenyl), 7.44 (d, 2H, m-phenyl).

9988 N-Oxide Formation and Decomposition by P450

CH3 I I

RPh-N : O=Fev

SCHEME 2. Possible pathways of deoxygenation of N,N-dimethylaniline N-oxides catalyzed by ferric P450.

RPh-N+--d I CH3

N-Methyl-2-aminoflwrene"-Aminofluorene (0.86 g, 4.7 mmol) was heated with CHII (0.80 g, 0.38 ml, 5.6 mmol) and NaHC03 (0.59 g, 7.0 mmol) in (CH3),C0 while stirring under reflux for 5 h. The suspension was filtered through paper and the resulting brown solution was dried in uacuo, dissolved in CHCl3, and washed with 1 N aqueous NaOH. The organic layer was dried with Na2S04, concentrated in uacuo, and applied to a silicic acid column (2.2 X 12 cm) equilibrated with CHCl,. The product of interest was eluted with 100 ml of CHC1,; this fraction was dried in uacuo, dissolved in CH30H, and further purified by preparative HPLC (10 X 250-mm Ultremex 5 octadecylsilane, 5 pm, Phenomenex) using a mixture of CHIOH and 25 mM phosphate buffer (adjusted to pH 7.0 with (C2H5),N) (92:8, v/ v) and a flow rate of 4.0 ml min". The N-methyl-2-aminofluorene fraction eluted at 5-6.5 min and was evaporated to dryness; the resulting precipitate was washed with alkaline aqueous CHIOH and the product was extracted into CH,Cl,. Evaporation of the solvent yielded the desired product, which was collected and dried in uacuo: m.p. 69-70 "C; 'H NMR ('H20) 6 2.91 (s, 3H, N-CHI), 3.82 (s, 2H, 9- H), 6.67 (d, lH), 7.17 (t, lH), 7.30 (t, lH), 7.45 (d, lH), 7.61 (t, 2H). N-Phenyl-2-pyrroline-N-Phenyl-2-pyrrolidone (Aldrich) was

treated with a limiting amount (1.2 hydride equivalents) of LiAlH4 in (C,H,),O at 0 "C according to Wittig and Sommer (1955); the product crystallized from CH30H and a portion was recrystallized from ab- solute C2H50H and dried in vacuo: m.p. 166.5-167.5 "C ((literature) 166-167.5 "C (Wittig and Sommer, 1955)); FAB+ MS: m/z 146 ([M + HI+, loo), dimer at m/z 289; UV: cyclohexane-X,, 251 nm, e251 18.0 mM" cm", X,, 300 nm, c300 4.0 mM" cm" (Wittig and Sommer, 1955); CH30H-X,, 251 nm, 6251 19.0 mM" cm", Xmax 307 nm, e307 4.1 mM" cm"; 1 N HCl-X,, 248 nm, c z a 10.3 mM" cm"; 'H NMR (C'HCl,) 6 3.28 (m, 2H, H-4), 3.43 (m, 2H, H-5), 5.10 (m, lH, H-3, J = 8.9 Hz), 6.40 (dd. lH, H-2). 6.82 (m, 3H, m- andp-phenyl), 7.28 (m, . .

2H, o-phenyl). . . ~

N.N.N-Trimethvl-4-methvlaniline-N.N-Dimethvl-4-methylaniline (i,N-dimethyi-p-toluidi"ne, 2.7 g, 20 mmol, dist:lled unde; aspi- rator vacuum) was heated with dimethyl sulfate (20 mmol) at 60- 70 "C, with stirring, for 6 h under a drying tube and then stirred for another 40 h at room temperature (Traber and Karrer, 1958). The product crystallized and was further precipitated by the addition of (C,H&O and was washed with (c2&)&: yield of N,N,N-trimethyl- 4-methylaniline methylsulfate 3.1 g (59%); m.p. 132-136 "C; FAB+- MS: m/z 150 (M+, loo), 135 ("15, 95), 120 ("30, 80); 'H NMR

SOzCH3), 7.47 (d, 2H, m-phenyl, J = 8.9 Hz), 7.71 (d, ZH, o-phenyl, J = 8.9 Hz).

N-Oxides were prepared according to the general procedure of Craig and Purushothaman (1970) with slight modification. m-Chlo- roperbenzoic acid was purified (from commercial 85% material) by washing a CH2C12 solution with 0.15 M phosphate buffer (pH 7.5) and drying in uacuo. A solution of 20 mmol of m-chloroperbenzoic acid (in 10 ml of CHCl,) was added gradually to 100 ml of an ice-cold stirred solution of 20 mmol of the N,N-dialkylaniline in CHC13.

('H20) 6 2.41 (s, 3H, PhCH,), 3.63 (5, 9H, -N(CH3)3), 4.81 (s, 3H, -

CH2OH I I CH3

RPh-N: Fe&t"-

RPhNHCHa + HCHO

CHyH

I RPh-N t

Stirring was continued for 3 h, during which time the mixture was allowed to come to room temperature. In all N-oxide syntheses, analytical TLC of the reaction mixture showed the production of N - oxide and presence of small amounts of unreacted parent amine. Each solution was applied to a column of basic alumina (2.2 X 27 cm) equilibrated with CHC1, and the column was washed with 500 ml of CHC13. N-Oxides were eluted with 250 ml of a mixture of CHCl3/ CH3OH (3:1, v/v). Analytical TLC of this fraction showed only one UV spot, one spot with tetracyanoethylene reagent (Damani et al., 1978), and no (UV) spot in the position of parent amine. N-Oxide fractions from alumina column chromatography were evaporated to dryness in vacuo; an equimolar amount of HC1 was added, and the sample was evaporated to dryness again. In the case of N-phenylpyrrolidine N-oxide, the fraction recovered from the alumina column was further purified by preparative TLC (Baker silica gel, CHC13/ CH30H/(C2H&NH (12:1:1, v/v/v), RF 0.54, as detected with tetracyanoethylene reagent (Damani et al., 1978)). The N-oxide was eluted from the silica with CH30H with the usual workup to give the HCI salt. HCl salts of N-oxides were recrystallized from the following solvent systems: (i) (CH&CO: N,N-dimethylaniline N-oxide, m.p. 121-122 "C; FAB+-MS: m/z 138 ([M + HI+, loo), 121 (M-16,66); 'H NMR (*H20) 6 3.88 (s, 6H, CH3), 7.58 (m, 3H, m- andp-phenyl), 7.87 (m, 2H, o-phenyl); N-phenylpyrrolidine N-oxide, m.p. 108-109 "C; FAB+-MS: m/z 164 ([M + HI+, 64), 147 ("16,100); 'H NMR ('HZO) 6 2.39 (m, 4H, NCH2CH2), 4.12 (m, 4H, N-CH,), 7.60 (m, 3H, m- and p-phenyl), 7.82 (d, 2H, o-phenyl); (ii) (CH3),CO/n-hexane: 4-methyl- N,N-dimethylaniline N-oxide, m.p. 124-125 "C; FAB+-MS: m/z 152

CH3), 3.88 (s, 6H, N-CH3), 7.46 (d, 2H, m-phenyl), 7.75 (d, 2H, o- phenyl); N,N-diethylaniline N-oxide, m.p. 135-137 "C; FAB+-MS: m/

CH,CH,), 4.11 (m, 4H, CH,CH3), 7.65 (m, IH, m- andp-phenyl), 7.75 (d, 2H, o-phenyl); N-methyl-N-ethylaniline N-oxide, m.p. 120- 121 "C; FAB+-MS: m/z 152 ([M + HI', loo), 135 (M-16,99); 'H NMR ('H20) 6 1.19 (t, IH, -CH,CH3), 3.78 (s, 3H, N-CH,), 4.01 (m, 2H, -NCH,CH3), 7.61 (m, IH, m- and p-phenyl), 7.80 (d, 2H, o-phenyl)); (iii) (CH3),CO/CH30H: 4-cyano-N,N-dimethylaniline N-oxide, m.p. 159-160 "C, FAB+-MS: m/z 163 ([M + HI+, 1001, 146 (M-16,56); 'H NMR ('H20) 6 3.89 (s, 6H, N-CHI), 8.07 (m, 4H, o- and m-phenyl). N,N-Dimethyl-2-aminofluorene N-oxide crystallized from

(CH3)&O/n-hexane as the zwitterion without added HCI; m.p. 140- 141 "C; FAB+-MS: m/z 226 ([M + HI+, 84) 209 (100); 'H NMR ('HzO) 6 3.67 (s, 2H, 9-H), 3.97 (s, 6H, N-CH3), 7.38 (m, 2H), 7.58 (d, 1H), 7.82 (q, 3H), 8.34 (s, 1H).

([M + HI+, go), 135 ("16, 100); 'H NMR ('HzO) 6 2.41 (5, 3H, 4-

z 166 ([M + HI+, loo), 149 (M-16,31); 'H NMR ('HzO) 6 1.19 (t, 6H,

Enzymatic Reactions For the measurement of rates of N-dealkylation andN-oxygenation

of the N,N-dialkylarylamines, equimolar amounts of rat liver P450 2B1 (Guengerich and Martin, 1980), rabbit NADPH-P450 reductase (Yasukochi and Masters, 1976), and ~-a-1,2-dilauroyl-sn-glycero-3-

N-Oxide Formation and

phosphocholine (to give a 30 p~ final concentration) (Guengerich, 1989b) were mixed together and preincubated for 5 min at 23 "C. (In some experiments, rabbit liver P450 1A2, prepared by the basic procedure of Alterman and Dowgii (1990) was used in place of P450 2B1.) The samples were then diluted in a mixture of 50 mM potassium phosphate buffer (pH 7.7) containing 5 mM MgCl,, 10 mM glucose 6- phosphate, 1 unit of glucose-6-phosphate dehydrogenase ml-', and the substrate. N,N-Dialkylarylamines were added at concentrations of 0.5-1.0 mM; the K,,, of rat P450 2B1 for N,N-dimethylaniline is 45 p~ (Okazaki and Guengerich, 1993). After shaking at 37 "C for 5 min to equilibrate the temperature, the last addition was NADP+ (final concentration 0.5 mM) to initiate NADPH production. When N-oxide formation was being measured, incubations were also done in the presence of bovine liver catalase (dialyzed before use to remove thymol, 100 units ml-l). Reactions were generally terminated by addition of one-third volume of 17% HClO, (w/v) and chilled on ice to precipitate phosphate salts. After centrifugation at 3 X lo3 X g for 5 min, the resulting supernatants were used for assays of N-dealkylation and N-oxygenation products (Scheme 3). Incubations were done in triplicate; control reactions included all components except the NADPH generating system and the values obtained were subtracted from those obtained in the presence of all components. In the case of the N-oxygenation rates, the values reported were those measured in the presence of catalase (this amount has previously been shown to remove all HzOz formed in such reactions (Guengerich and Strickland, 1977; Guengerich, 1978; Bondon et QL, 1989)). Experiments on the fates of N,N-dialkylaniline N-oxides were done in the same way except that in some of the studies the reactions of interest were done in the absence of NADPH and NADPH-P450 reductase and the control incubations contained all components except P450.

TLC The progress of organic synthesis and substrate purification was

monitored by TLC. Silica gel (Kodak) was used as the stationary phase. The solvent systems n-hexane/(CzHs),NH (99:1, v/v), n-hexane/CHCl3/(CZH5),NH (5:4:1, v/v/v), and CHCl,/CH,OH/ (CzH5)zNH (12:1:1, v/v/v) were used for substrate purification, 4-(N- methy1amino)benzonitrile synthesis and N-oxide synthesis, respectively. Amines and amine N-oxides were detected on TLC plates with UV light (using plates impregnated with fluorescent indicator) and N-oxides were also detected by color reaction after spraying with 0.5% (w/v) tetracyanoethylene in ethyl acetate (Damani et ~ l . , 1978).

HPLC HPLC was done with a Spectra-Physics 8700 pumping system

(Spectra-Physics, Piscataway, NJ) equipped with an Altex 210A injector (Beckman Instruments) and variable wavelength detector (LDC Spectromonitor 3100, Milton Roy, Riviera Beach, FL). Typi- cally the injector loop volume was 20 pl for analytical samples and 250 ~1 for preparative samples. Quantitation was generally done with

SCHEME 3. Flow chart for analytic procedures used in the analysis of N-oxygenation reactions catalyzed by P450 2B1. See "Experimental Pro- cedures" for details. "This method was used only in the measurement of N,N- dimethyl-2-aminofluorene N-oxide. bThis procedure was used to measure formation of 4-cyano-N,N-dimethylaniline N-oxide (UV detection, recovered from aqueous phase after CH,Cl, extraction, no Sep-Pak extraction used) and N,N-dimethyl[ring-"Claniline N-oxide (radiometric detection). 'This procedure was used in all other cases.

Decomposition by P450 9989

the use of internal standards and peak height analysis. HPLC octadecylsilane columns used included the following, Inertsil ODS-2 (5 pm, 4.6 X 150 mm, G& Science Inc., Tokyo, Japan), YMC-PACK ODS AM302 S-5 (120 A, 4.6 X 150 mm, Yamamura Chemical Lab., Kyoto, Japan), TSK-Gel ODs-BOT and ODs-12OT (4.6 X 150 mm, Tosoh, Tokyo, Japan), Ultracarb 5 ODs (30) (4.6 X 150 mm, Phen- omenex), Zorbax ODS (3 pm, 6.2 X 80 mm, Mac-Modd, Chadds Ford, PA), Beckman Ultrasphere ODS (5 pm, 10 X 250 mm, Beckman), and Ultremex 5 C18 (5 pm, 10 X 250 mm, Phenomenex). (C,H&N was distilled before use in HPLC solvents.

All of the arylamines and their N-alkyl and N,N-dialkyl derivatives were extracted quantitatively at alkaline pH. The general procedure is as follows. To 2.0 ml of each acidified enzyme reaction mixture was added the internal standard and 0.32 ml of 5 N NaOH; the product of interest was extracted (twice) into CHzClz. All of these amines were quantitatively extracted into the organic phase (results not shown). The internal standards used were N-ethylaniline (final concentration 100 p ~ ) for N,N-dimethylaniline N-demethylation and N,N-dimethylaniline (final concentration 100 p M ) for the other N,N- dialkylanilines, except 4-methyl-N,N-dimethylaniline for the assay of N,N-dimethylaniline recovered from the TiCl, reduction of its N - oxide derivative. In each case, the organic layer was dried with Na2S04 and evaporated to dryness under Nz at 40 "C in the presence of 100 pl of 0.1 M aqueous H3P04, which was shown to prevent any loss of amine. The dried samples were dissolved in 200 pl of a mixture of CH,OH/H,O (l:l, v/v) containing 75 mM (C2H5)3N. An aliquot was injected onto the HPLC column; the mobile phase consisted of a mixture of CH30H and buffer (25 mM H3P04, adjusted to pH 7.0 with(CzH&N) (Fig. 1). The ratio of CH3OH to buffer in mobile phase was adjusted to elute the product of interest with t R -10 min (Table I).

The N-dealkylation of N-phenylpyrrolidine was assayed in the following manner. Incubations were terminated by the addition of 0.2 ml of 6 N KOH (to a 1.0-ml incubation volume); the imine formed by N-dealkylation was thus converted to N-phenyl-2-pyrroline. The product was extracted twice with 2 volumes of (C,H&O, and the combined organic phases were dried with MgSO, and concentrated to dryness under a stream of Nz at 23 "C. Samples were then dissolved in 200 pl of a 1:l (v/v) mixture of CH3OH and 50 mM H3P04 adjusted to pH 2.5 with (CzHJ3N; 20 pl of each sample were injected onto a 10 x 250-mm Beckman Ultrasphere ODS HPLC column. The column was eluted with a solvent consisting of a 9:l mixture (v/v) of CH,OH and 50 mM HaPo4 adjusted to pH 2.5 with (C2H5),N. The flow rate was 4.0 ml min" and the effluent was monitored at 254 nm. Under these conditions N-phenylpyrrolidine eluted at t R 6.0 min and N - phenyl-2-pyrroline eluted at t R 14.8 min. In this case quantitation was done with external standards.

In the assay of N-oxide formation, 2.0 ml of acidified microsomal reaction mixture was made alkaline by addition of 0.32 ml of 5 N NaOH and applied to a Sep-Pak C,S cartridge equilibrated with H,O (after previous washing with CH30H). After washing the cartridge

Pa50 Reaction HC104 Centrifugation

Supernatant

1 AddNaoH

J"""$"i"n /\ Sep-pak Extraction,

CH2C12 Phase Aqueous Phase - -. i /\

7\ "i HPLC Prep. HPLC

Preparative Analytical Colorimetric Assay (Amines, ITiCI, Reduction HPLC HPLC some Noxidesg I (N-dealkyation)

HPLC (of NOxidesc) 1 nc13 Extraction

Analytical HPLC.


with an aqueous solution (5 ml of H20 for N,N-dimethylaniline N- oxide, 4-methyl-N,N-dimethylaniline N-oxide, and N-methyl-N- ethylaniline N-oxide analysis, 2 ml of H20 for 4-cyano-N,N-dimethylaniline N-oxide analysis, or 5 ml of 5% aqueous CH30H solution (v/v) for N,N-diethylaniline N-oxide analysis), the N-oxide was eluted with 4 ml of an aqueous CH30H solution (20% CH30H (v/v) for 4-cyano-N,N-dimethylaniline N-oxide, 30% CH30H(v/v) for N,N-dimethylaniline N-oxide, 50% CH30H (v/v) for N-methyl-N- ethylaniline N-oxide, and 60% CH30H (v/v) for 4-methyl-N,N-dimethylaniline N-oxide and N,N-diethylaniline N-oxide). The eluted N-oxide fraction was added to 100 pl of 1 M aqueous H3P04 and evaporated to dryness under N2 at 40 "C. Each sample was then dissolved in 100 pl of 1 N aqueous NaOH and 50 pl was used for HPLC. The mobile phase was as in the case of the other N,N- dimethylaniline oxidation products, with the ratio of CH30H to buffer chosen such that the tR of the most slowly eluted metabolite was -7 min (flow rate was 1.5 ml rnin"). We found that the different octadecylsilane columns varied considerably in separation efficiencies for the various N,N-dimethylaniline N-oxides. The most efficient columns of those examined were the Inertsil ODS-2 (1200-2700 theoretical plates for the N-oxides) and Ultremex 5 ODS (800-2100 theoretical plates for the N-oxides). The poorest of the columns (see above), even when new, had plate numbers in the range of 120-510.

With the low concentrations of N-oxides analyzed in reaction mixtures, four basic procedures were performed, depending upon the substrate and the properties of the product, as described below (Scheme 3).

(i) When N,N-dimethyl[ring-'4CC]aniline (1.1 mCi mmol") was used as substrate, the N-oxide produced was concentrated by the Sep- Pak procedure (Scheme 31, separated by HPLC, and quantitated by measurement of radioactivity in the HPLC eluate (3 ml of eluate added to 15 ml of Scinti-Verse I1 scintillation mixture) using a Beckman LS 7000 liquid scintillation counter (Beckman).

(ii) In the case of the substrate 4-cyano-N,N-dimethylaniline, 2.0 ml of the acidic supernatant recovered from HC1O4 treatment of the reaction was mixed with 0.32 ml of 5 N aqueous NaOH and the solution was washed 3 times with CH2C12. The resulting aqueous phase was acidified by the addition of 150 pl of 5 N HC1 and treated with 120 pl of the 30% (w/v) TIC13 reagent (dissolved in 5 N HCl) to reduce the N-oxide to the amine. After 1 h the solution was made alkaline by the addition of 5 N NaOH, the internal standard N,N- dimethylaniline was added, and the internal standard and 4-cyano- N,N-dimethylaniline were extracted into CH2C12 and analyzed by HPLC as described above, using UV detection.

(iii) When the substrate was N,N-dimethyl-2-aminofluorene, 2.0 ml of the acidic supernatant derived from treatment of the reaction mixture with HClO, were mixed with 0.32 ml of 5 N aqueous NaOH and the N-oxide was quantitatively extracted into CHZCI, (2 extrac- tions). The organic phases were combined, dried with Na2S0, and reduced to dryness under N2 at 40 "C after the addition of 100 pl of 0.1 M aqueous H3P0,. The sample was dissolved in 200 pl of 50% (v/ v) aqueous CH30H containing 75 mM (C2Hd3N and injected onto a 10 X 250-mm Ultremex 5 CIS HPLC column. A mobile phase consisting of CH30H and 25 mM phosphate buffer (adjusted to pH 7.0 by the addition of (C2H,)3N (90:10, v/v)) was used to elute the column at a flow rate of 3.0 ml rnin". The fraction corresponding to the elution time of standard N,N-dimethyl-2-aminofluorene N-oxide (5.5-6.8 min) was collected, mixed with 20 pl of 5 N aqueous HC1, concentrated to -2 ml under an N2 stream at 40 "C, and treated with 120 pl of the 30% Tic13 reagent (w/v, in 5 N aqueous HC1) for 1 h. The solution was then raised to alkaline pH by the addition of 5 N aqueous NaOH, and the resulting N,N-dimethyl-2-aminofluorene was extracted into CHZClz and analyzed by HPLC.

(iv) With the other amines as substrates, the N-oxide fraction was isolated from 5.0 ml of acidified microsomal reaction mixture by solid phase extraction as described above (Scheme 3). The Sep-Pak eluate was concentrated to a volume of 250 pl, and 200 pl of each sample were used for preparative HPLC (10 X 250-mm Ultremex 5 C,S) using a mixture of CH30H and buffer (25 mM &Po& adjusted to pH 7.0 with (C2H&N). The ratio of CH30H to buffer in the mobile phase was chosen so that the tR of the N-oxide was -7 min (flow rate 4.0 ml rnin"). The N-oxide fraction of the HPLC eluate was acidified by the addition of 50 p1 of 5 N aqueous HCl, concentrated to -2 ml under N, a t 40 "C, and treated with 120 pl of 30% Tic13 solution (w/ v, dissolved in 5 N aqueous HCl) for 1 h. TiCl, quantitatively reduced all of the N-oxides to the parent amines, thus increasing the absorbance considerably (Table I). After the addition of 200 p1 of 5 N aqueous NaOH and the appropriate internal standard, the parent

amine was extracted into CH2C12, concentrated, and analyzed by HPLC as described above, using UV detection.

Colorimetric Assay of N-Oxides N,N-Dimethylaniline N-oxide was quantitated in some cases by

the colorimetric method of Ziegler and Pettit (1964). After acidifica- tion of the enzyme incubation, the mixture was made alkaline by addition of NaOH and extracted twice with CH,Cl,. The combined organic phases were used for HPLC analysis. The aqueous phase was extracted once more with CHZC12 and a part of the aqueous phase was used for colorimetric analysis. A standard curve was prepared by adding known amounts of the N-oxide to the microsomal reaction mixture (zero reaction time); linearity was observed in the N-oxide concentration range of 5-100 nmol. As pointed out under ''Results" (see below), we found that this approach was not very sensitive nor discriminating for the enzyme assays but was often used in the development of analytical procedures.

Spectroscopy and Other Assays Aniline derivatives were generally dissolved in a mixture of CH30H

and buffer (25 mM H3P04 adjusted pH 7.0 with (C2H5)3N (6040, v/ v)) unless otherwise noted and UV spectra were recorded on a Cary 210 spectrophotometer in the automatic base-line correction mode (Varian, Walnut Creek, CA) or a Cary 14/0LIS system (OLIS, Bogart, GA). 'H NMR spectra were recorded on a Bruker AM-300 spectrometer (300 MHz, Bruker, Billerica, MA). N-Oxides were dissolved in 'H20 and sodium 3-(trimethylsilyl)-[2,2,3,3-2H4]pr~pionate was used as the internal standard. Other amines were dissolved in C'HCl, and (CH3),Si was used as the internal standard. Fast atom bombardment-mass spectra were recorded in the Vanderbilt facility by B. Nobes on a VG 70-250 instrument having extended geometry, a standard VG FAB ion source, an Ion-Tech saddle field FAB gun producing Xe atoms of 8 kV energy, and a VG 11/250 data system (VG, Manchester, United Kingdom). Glycerol was used as the matrix.

Melting points were obtained using a Mel-Temp apparatus (Mel- Temp, Cambridge, MA) and are uncorrected. P450 concentrations were measured using the procedure of Omura and Sato (1964).

RESULTS

Assay Development-While rates of N,N-dimethylaniline N-oxide formation and reduction are rapid in liver microsomes, our preliminary studies indicated that much of these reactions could be attributed to enzymes other than P450 and that simple colorimetric assays (Ziegler and Pettit, 1964) and direct HPLC would not be sensitive nor specific enough. HPLC analysis is not trivial because of the hydrophilic nature of these compounds and their low UV extinction relative t o the anilines (Table I). We also realized that without the use of radioisotopes for all substrates, development of methods for extraction and concentration would also be necessary. Conditions were developed for absorption to and elution from octadecylsilane cartridges for each of the N-oxides of interest (see "Experimental Procedures"); the recovery was >95% under all of the conditions utilized here, except for 4-cyano- N,N-dimethylaniline N-oxide (70%). The effluent eluting with the appropriate tR was collected and reduced to the amine with T ic& (Gorrod, 1981) (Fig. 1). Under the conditions used, the reduction of all N-oxides considered here was essentially quantitative and linear standard curves were constructed in the range of 0.1-15 nmol. I t should also be emphasized that all of the substrates, both N,N-dialkylanilines and their N- oxides, were rigorously purified and analyzed before use. The low levels of product formation seen required such purity in order to suppress background levels in the assays.

Fates of N-Oxides-There is precedent for the NADPH- dependent reduction of N,N-dialkylaniline N-oxides to the corresponding N,N-dialkylanilines by P450s (Sugiura et al., 1976; Iwasaki et al., 1977).

PhNR2-0 PhNR2 (Eq. 1)

However, we found that the rates measured with P450 2B1 in

N-Oxide Formation and Decomposition by P450 9991

TABLE I Spectrat and HPLC characteristics of compounds used in this study

t R

Compound UV A,,, nm System 1" System

2*

loglo E , M" em" min

Aniline 227 (3.75), 280 (3.17) 2.6 8.8 N-Methylaniline 240 (4.03), 286 (3.34) 3.6 N-Ethylaniline 240 (4.03), 286 (3.32) 4.1 N,N-Dimethylaniline 248 (4.07), 288 (3.31) 6.9 N-Ethyl-N-methylaniline 250 (4.04), 293 (3.17) 10.0 N,N-Diethylaniline 258 (4.15), 302 (3.41) 15.6 4-Methylaniline 229 (3.751, 285 (3.15) 3.1 4-Methyl-N-methylaniline 239 (3.97), 292 (3.16) 4.9 4-Methyl-N,N-dimethylaniline 244 (4.12), 288 (3.30) 10.2 4-Aminobenzonitrile (4-cyanoaniline) 255 (3.64), 282 (3.61) 2.2 4-Cyano-N-methylaniline 267 (366), 306 (3.62) 3.0 4-Cyano-N,N-dimethylaniline 297 (4.37) 4.3 N,N-Dimethylaniline N-oxide 253 (2.40) N-Ethyl-N-methylaniline N-oxide 254 (2.62) N,N-Diethylaniline N-oxide 254 (2.38) 4-Methyl-N,N-dimethylaniline N-oxide 252 (2.42), 257 (2.44) 4-Cyano-N,N-dimethylaniline N-oxide 269 (2.53), 276 (2.88) N-Phenylpyrrolidine' 253 (4.15), 302 (3.27) N-Phenyl-2-pyrroline' 251 (4.28) N-Phenylpyrrolidine N-oxide' 255 (2.73) 2-Aminofluorene' 281 (4.35) N-Methyl-2-aminofluorene' 291 (4.29) N,N-Dimethy1-2-aminofluorenec 295 (4.39) N,N-Dimethyl-2-aminofluorene N-oxide' 264 (4.31), 288 (3.88), 299 (3.95)

10.3

3.3 6.2

12.1 8.7 2.4

a TSK ODs-12OT octadecylsilane column (4.6 X 150 mm), CH30H, 25 mM H3P04/(CZH5)3N (pH 7.0) (6535, v/v), flow rate 1.0 ml rnin".

e See "Experimental Procedures." Same column as System 1, CH30H, 25 mM H3PO,/(CZH5),N (pH 7.0) (15:85, v/v), flow rate 1.5 ml rnin".

0 2 4 0 0 10 12 14 16 100 2 4 S 8 10 12 14

Tlme, rnin FIG. 1. HPLC of products of 4-methyl-N,N-dimethylaniline

oxidation by P460 2B1. 4-Methyl-N,N-dimethylaniline was in- cubated with the reconstituted P450 system in the manner described under "Experimental Procedures," with the addition of catalase (5 ml of incubation volume, 0.3 WM P450, reaction time 5 min). Solid phase extraction was used to concentrate the desired 4-methyl-N,N-dimethylaniline N-oxide fraction and this was injected onto a 10 X 250-mm Ultremex 5 Cls HPLC column (part A ) . The column was eluted with a 35:65 (v/v) mixture of CH30H and 25 mM phosphate buffer (adjusted to pH 7.0 with (C,H&N), using a flow rate of 4.0 ml rnin". The efluent corresponding in tR to the N-oxide (shown by hatched rectangle) was treated with Tic13 and the product was extracted into CHZCIZ at alkaline pH after the addition of the internal standard N,N-dimethylaniline. This material was injected onto an analytical Ultracarb 5 ODS (30) column (4.6 X 250 mm) (part B ) and eluted with a 75:25 (v/v) mixture of CH30H and the buffer described above. The positions of the eluted compounds are shown in both parts of the figure. The bars show the absorbance scales for the two different traces in each part.

the presence of NADPH-P450 reductase (Table 11, last column) were considerably less than those estimated in microsomes, by about 1 order of magnitude (results not shown). Thus, not all of the microsomal reduction may be attributable to P450s (Lashmet-Johnson and Ziegler, 1986). The rates

were difficult to measure accurately due to the presence of trace residual N,N-dimethylanilines; no clear trend associated with electron-donating capability was seen among the N,N- dimethylanilines and (somewhat surprisingly) we could not demonstrate reduction of the N-ethyl-substituted anilines. Rates of NADPH-dependent conversion of N,N-dialkylaniline N-oxides to N,N-dialkylaniline products were higher (Table 11) and N-demethylation appeared to be favored over N-deethylation.

As reported previously by our laboratory (Burka et al., 1985) and others (Heimbrook et al., 1984), P450s can also catalyze the slow NADPH-independent decomposition of N,N-dialkylaniline N-oxides (Table 11).

PhN(CH,R),-0 = PhNHCH,R + RCHO (Eq. 2)

N-Dealkylation products were found with all N,N-dialkylaniline N-oxides examined and N-demethylation was favored over N-deethylation in the case of N-ethyl-N-methylaniline. However, deoxygenation of N,N-dialkylaniline N-oxides

PhN(CH,R),-0 = PhN(CHzR)2 0%. 3)

appeared to be very unfavorable in the absence of NADPH, with only a suggestion of N,N-dialkylaniline formation in one case ( i e . N-ethyl-N-methylaniline N-oxide). In the N-dealkylation reactions there did not appear to be a clear pattern in terms of electron-donating capability.

We previously reported that N,N-dialkylaniline N-oxides were incapable of supporting P450-dependent aminopyrine N-demethylation at rates >2 nmol of product formed min" (nmol of P450)" (Burka et al., 1985). We considered the possibility that a more favorable protocol for demonstrating N,N-dimethylaniline N-oxide-dependent oxidation by P450 2B1 might involve the use of N,N-dirnethyl[rir~g-'~C]aniline


TABLE I1 N-Oxide transformation by P450 2Bl/NADPH-P450 reductase system

All rate estimates are means k S.D. (n = 3). N-Demethylation product N-Deethylation product Reduction product

Substrate (monoalkylaniline) (monoalkylaniline) (N,N-dialkylaniline) -NADPH +NADPH -NADPH +NADPH -NADPH +NADPH

nmol product formed min" nmol product formed min" (nrnol P450)"

nmol product formed min" (nmol P450)-' (nmol P450)"

N,N-Dimethylaniline N-oxide 0.83 k 0.01 2.58 f 0.05 4-Methyl-N,N-dimethylaniline 0.29 -+ 0.10 1.78 f 0.05

-0.27 f 0.14" 0.21 f 0.02 -0.10 f 0.01" 0.92 f 0.04

4-Cyano-N,N-dimethylaniline C0.05 0.88 zk 0.31 -0.89 f 0.15" 1.74 f 0.62

N,N-Diethylaniline N-oxide 0.023 f 0.008 0.41 f 0.04 0.048 k 0.040 -0.51 f 0.02" N-Ethyl-N-methylaniline N-ox- 0.69 f 0.05 3.59 f 0.11 0.17 f 0.04 0.43 f 0.02 0.43 f 0.24 -3.25 f 0.12"

N-oxide

N-oxide

ide In these cases the recovery of the N,N-dialkylaniline was actually somewhat less in the presence of the P450 than in the absence. The

result is due to the loss of the traces of N,N-dialkylaniline in the N-oxide peparations.

as a substrate and HPLC assay of the formation of N- methyl[ring-"Claniline.

R"PhN(CH2R)z-0 + *PhN(CH& *PhNHCH3 (Eq. 4)

Thus, if N,N-dimethylaniline N-oxide were to transfer its oxygen to ferric P450, the product N,N-dimethylaniline might be expected to rapidly equilibrate with the 14C-labeled substrate. However, none of the above three N,N-dimethylaniline N-oxides (see above) was found to be capable of supporting the N,N-dimethyl[ring-'4CJaniline N-demethylation reaction at a rate of 32 0.4 nmol of product formed min" (nmol of P450)" (the value of 0.4 min" was measured with 4-cyano- N,N-dimethylaniline N-oxide; all other oxides gave rates of (0.05 min"). For comparison, this rate (0.4 min") is 1% of that measured in the NADPH-dependent reaction (see above) and 0.1% that measured in the reaction supported by the oxygen surrogate iodosylbenzene (Macdonald et al., 1989).

Another aspect considered was the possible inhibition of P450 2B1 reactions by N-oxides. The aminium radical mechanism (Scheme 1) involves development of a positive charge in the transition state, and we considered the possibility that N,N-dimethylaniline N-oxides and other positively charged analogs might behave as transition state analogs. However, the rate of 0-dealkylation of 7-pentoxyresorufin by rat liver microsomes (4.6 nmol of product formed by min" [mg of protein]-') was not decreased by 60-100 p~ concentrations of N,N-dimethylaniline N-oxide, 4-methyl-N,N-dimethylaniline N-oxide, 4-cyano-N,N-dimethylaniline N-oxide, or N,N- diethylaniline N-oxide or the quaternary amine 4-methyl- N,N,N-trimethylaniline. The N,N-dimethylanilines were actually better inhibitors of 7-pentoxyresorufin 0-dealkylation (20-90% inhibition in this concentration range). We also found that the N,N-dimethylaniline N-oxides did not inhibit the N-demethylation of N,N-dimethyl[ring-14C]aniline (<7% inhibition at concentrations of 0.2-2 mM).

Formation of N-Oxides-The development of appropriate preparative and analytical procedures for dealing with small amounts of the N-oxides under consideration has already been presented. Microsomal preparations generate considerable amounts of the N,N-dialkylaniline N-oxides, but most of these products are formed by the flavin-containing monooxygenases (Ziegler, 1991).

PhNR, a PhNR2-0 (Eq. 5)

Hlavica and Hulsmann (1979) have reported that rabbit P450 1A2 ("P448") could convert N,N-dimethylaniline to the

N-oxide at a rate of 1-2 nmol min" (nmol of P450)", but Pandey et al. (1989) could not detect a rate 20.3 nmol of product formed min" (nmol of P450)" with the same enzyme. We measured this reaction with this enzyme (rabbit P450 1A2) using N,N-dimethyl[ring-'4C]aniline as substrate and separated the N-oxide by HPLC after concentration with Sep-Pak cartridges. The rate of N-oxide formation was 0.0047 k 0.0014 nmol min" (nmol of P450)" (n = 3), which can be compared to an N-demethylation rate of 3.3 nmol min" (nmol of P450)", measured under exactly the same conditions (the ratio of the latter to the former is 720). Further studies were focused on (rat) P450 2B1 because the N-demethylation rate was known to be considerably higher (Okazaki and Guenger- ich, 1993).

When P450 2B1 was examined in a reconstituted enzyme system, we found that N,N-dimethyl[~ing-'~C]aniline was converted to the N-oxide, as analyzed by HPLC (Table 111). Furthermore, an identical rate was obtained when the unlabeled substrate was used, the HPLC zone corresponding to the N-oxide was collected and reduced with TiC13, and the resulting product N,N-dimethylaniline was analyzed by HPLC.

In further control experiments, it was demonstrated that the substrate must be present during the incubation for the reaction to occur (and could not be added after termination in this procedure). Appreciable activity was not seen if either the P450 2B1 or NADPH-P450 reductase fraction was omit- ted from the (NADPH-fortified) incubation, arguing against the presence of trace levels of microsomal flavin-containing monooxygenase. We considered the possibility that adding an N-oxide might serve to help trap the same (radioactive) product by sparing its decomposition, but the presence of 0.5 mM N,N-dimethylaniline N-oxide in an incubation did not affect the level of radioactive N-oxide formed from N,N- dimethylaniline. The addition of either superoxide dismutase (50 pg ml-l) or glutathione (5 mM) did not significantly alter the level of N,N-dimethylaniline N-oxide formed, arguing against roles for superoxide anion and hydroperoxides in the system. Lipid hydroperoxides would not have been added to the system because the only lipid used was L-a-1,Z-dilauroyl- sn-glycero-3-phosphocholine. Detergents are used in the purification of P450 and NADPH-P450 reductase and could conceivably be a source of hydroperoxides. We have previously demonstrated that the level of nonionic detergent associated with these enzymes to be t 2 nmol of monomer (nmol of P450)" and the level of cholate and phospholipid to be <1 nmol (nmol of P450)" (Muller-Enoch et al., 1984). When a


TABLE 111 Rates of N-dealkylation and N-oxygenation of N,N-dialkylaniline derivatives by P450 2Bl

All rate estimates are presented as means f S.D. ( n = 3) (after subtraction of blank, minus enzyme/complete incubation at zero time, in each case). Catalase was present in all assays for which results are presented here.

Substrate Rate

N-Dealkylation Ratio

N-Oxygenation

nmol product formed min” (nmol P450)”

N,N-Dimethylaniline 34.7 f 0.2 0.040 f 0.008 940 4-Methyl-N,N-dimethylaniline 39.8 f 3.1 0.097 f 0.017 400 4-Cyano-N,N-dimethylaniline 18.0 f 1.9 0.183 f 0.087 98 N-Ethyl-N-methylaniline 42.7 f 2.2 (demethylation) 0.051 f 0.006 890

2.7 f 0.2 (deethylation) 0.025 f. 0.004 1020 0.007 f. 0.005 1100

N,N-Diethylaniline 25.5 f 0.1 [*Hl0]N,N-Diethylaniline 7.9 f 0.2 N-Phenylpyrrolidine 8.2 f 1.0 N,N-Dimethyl-2-aminofluorene 3.1 f 0.7

10-fold molar excess of the nonionic detergent, Emulgen 911 (routinely used in the purification of P450 2B1 and NADPH- P450 reductase), was added to an incubation containing N, N- dimethyl[ring-’4C]aniline, there was no significant change in the level of N,N-dimethylaniline N-oxide formed (50 nmol of detergent added to 5 nmol of enzyme in a volume of 2.0 ml).

The approach of isolation and TiC13 reduction was utilized to analyze rates of N-oxygenation of several other nonradioac- tive N,N-dialkylaniline derivatives, and rates of N-dealkylation were also measured in each case (Table 111). Comparison of the rates indicated that the ratio of N-dealkylation to N- oxygenation (Scheme 1) varied from 1020 to 98 within the basic series of N,N-dialkylanilines. In these reactions the presence of catalase did not alter the level of N-oxide formed in most cases; the values reported in Table I11 were all measured in the presence of what appears to be a sufficient level of catalase to destroy all HzOz produced (Guengerich and Strickland, 1977; Guengerich, 1978; Bondon et al., 1989). No clear trend was seen in the effects of para electron- donating substituents on the ratio of N-dealkylation to N- oxygenation. Although the N-methyl group is lost in prefer- ence to the N-ethyl group in the N-dealkylation of N-ethyl- N-methylaniline, the ratio of N-dealky1ation:N-oxygenation was not decreased in the case of N,N-diethylaniline.

N-Phenylpyrrolidine and N,N-dimethyl-2-aminofluorene can be defined as extended analogs of N,N-dimethylaniline and were examined. N-Phenylpyrrolidine was considered because some degrees of freedom of rotation of the ethyl groups are lost, possibly leading to a more favorable orbital overlap for oxygen rebound, and N,N-dimethyl-2-aminofluorene was considered because of the known N-oxygenation of %aminofluorene by P450 2B1 (Frederick et al., 1982). Special assay conditions had to be developed for the measurement of the products of these oxidation reactions (Scheme 3 and “Exper- imental Procedures”). N-Oxides were formed with both of these substrates (which may be considered derivatives of N,N- dimethylaniline). The ratios of N-demethy1ation:N-oxygenation observed were 140 and 6, respectively, for N-phenylpyrrolidine and N,N-dimethyl-2-aminofluorene.

DISCUSSION

A major finding presented in this article is that P450 enzymes form finite levels of N-oxides, even in instances where a-protons can be readily lost to yield N-dealkylation products. Demonstrating the formation of low levels of N- oxides was a technically challenging task, however, because of the physical characteristics of these compounds. N,N- Dimethyl[ring-’4C]aniline was used as the substrate and labeled N,N-dimethylaniline N-oxide was recovered after

0.057 k 0.018 140 0.49 f 0.06 6

HPLC. In the other approach, the problem of low UV absorbance of the N,N-dialkylaniline N-oxides was addressed by isolation of the N-oxide fraction (by HPLC), reduction to the amine (of higher UV absorbance) by TiC13, and further HPLC. The two approaches yielded similar estimates of rates when compared. I t should also be re-emphasized that (i) levels of N-oxides estimated in the various cases were significantly greater than the background levels, and (ii) the N-oxides were produced even in the presence of levels of catalase known to abolish H202 formation. This latter point is important in ruling out N-oxygenation by H20, produced in an uncoupled reaction. Varying the concentration of catalase was found not to alter the level of N-oxide formed from NJV-dimethylaniline. Furthermore, when levels of Hz02 shown to be formed in the P450 reaction were added (measured by colorimetric assay (Thurman et al., 1972) and the effect of catalase upon 0, uptake (Nordblom and Coon, 1977); results not shown), no N-oxides were produced in the cases examined. Therefore, we interpret our results to indicate that P450s catalyze the slow formation of N-oxides and probably hydroxylamines as a general feature of amine oxidation, even if it is a minor pathway relative to N-dealkylation. It should be emphasized again that, in general, the flavin-containing monooxygenases make a much greater contribution to the N-oxygenation reactions; the catalytic mechanism precludes direct N-dealkylation (Ziegler, 1991).

The mechanism in Scheme 1 is presented as the simplest paradigm that can explain all of the observed results, although some alternate possibilities such as direct oxygenation of the substrate cannot be unambiguously ruled out. The aminium radical, formed by 1-electron oxidation (Macdonald et al., 1989; Guengerich and Macdonald, 1990; Okazaki and Guen- gerich, 1993), is an intermediate and partitions between radical recombination (to yield the N-oxide or hydroxylamine) and a-hydrogen abstraction, followed by radical recombination, to yield the carbinolamine (and N-dealkylation products). We have recently presented evidence that the Fe02+ entity in P450s (but not peroxidases) can act as a specific base to facilitate the deprotonation step (Okazaki and Guen- gerich, 1993). The prominence of heteroatom oxygenation products in P450 reactions involving sulfur compounds (Wa- tanabe et ul., 1980, 1981, 1982a, 1982b; Fujimori et ul., 1986) is also consistent with the view (but does not in itself prove) that cation radicals are involved in the formation of N-oxides.

There are, however, some inherent problems with the N- oxygenation step in the reductionistic paradigm of Scheme 1. If direct recombination were operative and rapid, we would have expected the para substituents to influence rates of N- oxide formation in the same way as N-dealkylation (Burka et


al., 1985; Macdonald et al., 1989). However, this was not the case (Table 111). I t should be mentioned that Burstyn et al. (1991) did not see an effect of para substitution on rates of formation of N-hydroxyanilines. Aminium radicals lose methyl groups through N-dealkylation more rapidly than ethyl groups (Table 111); we considered the possibility that N,N-diethylaniline might give a higher ratio of N-oxide:dealkylated product but it did not. Kurebayashi (1989) had examined the deamination and N-hydroxylation of cy- clohexylamine in rabbit liver microsomes and was able to show a small shift towards the latter pathway upon deuterium substitution. We considered this possibility with N,N- diethylaniline; however, both reaction rates were apparently decreased and we could not demonstrate a significant change in the ratio (Table 111). We also considered 1-naphthylamine and its N,N-dimethyl derivative. The rate of N-demethylation of N,N-dimethyl-1-naphthylamine was 12 nmol of N-methyl- 1-naphthylamine formed min” (nmol of P450)” (Scheme 4). Although we did not make an extensive search for the N- oxide of N,N-dimethyl-1-naphthylamine, we had previously reported that 1-naphthylamine itself is not converted to the hydroxylamine (Hammons et al., 1985) (but does form the 2- hydroxy derivative). If the paradigm of N-oxygenation were as simple as electron abstraction and rebound, we would have expected the hydroxylamine to be formed in light of the apparent ease of electron abstraction in the N-demethylation of N,N-dimethyl-1-naphthylamine.

If the N-oxygenation process cannot be explained in terms of direct rebound to the aminium radical, what are other possibilities? First of all, the lack of u+ effect is probably not due to differences in the stabilities of the N-oxides, as consideration of Table I1 does not improve the analysis of Table 111. Although the formation of sulfur oxides by P450s has been postulated to involve direct oxygen transfer to a sulfur radical cation and a u+ effect with a negative p value has been reported, closer analysis indicates that the analysis was all based upon rates of NADPH oxidation, not product formation, and that these rates were relatively low and may well reflect formation of H202 and HzO (Watanabe et al., 1980, 1981, 1982a, 1982b; Fujimori et al., 1986). Thus, a clear para dependence for heteroatom oxygenation has not been demonstrated in the above references or other work (Plb and Marnett, 1989). One consideration is the acidity of the N-H

p45022 +HCHO

/ / k=12min ’ l / /

NH2 NH-OH I I

SCHEME 4. Oxidation ofN,N-dimethyl-1-naphthylamineand 1-naphthylamine by P450 2B1 (Hammons et al. (1985) and this work).

bond of an aminium radical (Scheme 5; for reference, see Bondon et al. (1989) and references therein). This may be a factor to consider in the general mechanism, although it is not relevant to the N,N-dialkyl compounds. Another possibility is a subsequent electron transfer from the aminium radical to a yield a cation/FeOH*+ pair that generates the product. The ease of oxidation of the “second” electron from an aminium radical is fairly well established in electrochem- istry (Weinberg and Brown, 1966; Smith and Mann, 1969; Masui and Sayo, 1971; Yasukochi et al., 1979; Shono, 1984) and the two potentials of the FeO complex are thought to be similar in the case of models (Lee et al., 1985; Groves and Gilbert, 1986; Hayashi and Yamazaki, 1979). Indeed, we previously reported that the oxidation products generated from 1-naphthylamine, 2-naphthylamine, and 2-aminofluorene by P450s were best modeled by Huckel calculations involving such FeOH*+/cationic nitrogen pairs, in the absence of con- founding steric restriction by the protein (Hammons et al., 1985).

Nevertheless, if only electron transfer reactions control rates of N-oxygenation, one might still expect to see a consistent para substituent effect. We postulate that what may be rate-limiting is the formation or decomposition of Fe. heteroatom oxide complexes. The formation could occur after either 1- or 2-electron oxidation (Scheme 6). The rates of decomposition of such complexes could be influenced by para substituents, and even the reduction of these complexes is probably possible in consideration of the low rates of product formation (see Tables I1 and 111). Thus the effects of para substitution might have counteracting effects and no clear trend might be seen. The Fe(II1). N-oxide complexes appear to be undetectable in the near UV/visible spectrum and the means to study the kinetics are not currently obvious. Never- theless, there does appear to be more to N-oxygenation than simple oxygen transfer to an aminium radical.*

In some cases, ratios of P450-catalyzed N-dealky1ation:N- oxygenation are relatively low, such as for the substrates N- (1-phenylcyclobuty1)benzylamine (-10) (Bondon et al., 19891, sencionine (-5) (Williams et al., 1989), and methamphetamine (2-14, depending upon the particular P450 enzyme) (Baba et al., 1987). Previously the low rates of N-oxygenation in the presence of accessible a-hydrogens had been over- looked; we have expressed the view that N-oxygenation only occurs in the absence of these a-hydrogens (Guengerich and Macdonald, 1984) and, at a gross level, this view is still correct. However, it now appears that there is always a finite partition ratio occurring between N-oxygenation and deprotonation followed by oxygen rebound to yield a carbinolamine (Scheme 1). The balance would appear to be a function of both the particular P450 enzyme and substrate. Situations in which P450s yield N-oxides, hydroxylamines, and nitrones (Wil- liams et al., 1989; Baba et al., 1987; Bondon et al., 1989) should not be considered special exceptions but rather parts of a continuum in which rates for the various steps are affected by aspects of the interaction of the heme moiety with the apoprotein and substrate in terms of individual transition states. Therefore it may be expected that more situations will be encountered in which P450 amine N-oxygenation products are seen.

* An alternate possibility that may be considered is that the N - oxides may derive from the Fe(II1). OOH complex prior to the usual 0-0 bond scission in the general P450 oxygen activation step. AI- though such a possibility cannot be unambiguously ruled out, there is no well documentated precedent for such reaction in the oxygenation of other substrates, all of which appear to proceed in reasonable yield from biomimetic models containing a single oxygen (e.& Mansuy et al., 1989).


SCHEME 5. Possible mechanisms involved in N-dealkylation (Oka- zaki and Guengerich, 1993) and in N-hydroxylation of primary of primary and secondary amines.

pKa - 9 $. " N H p + HCHO

1 a'

Fell' 0- I I

+ volved in N-oxygenation of tertiary

Fe"'

Phh+Me2 O=Fe" --f 0 Fe"' + PhNMe, - 1

PhNMe, SCHEME 6. Possible events in-

amines.

Fell' 0

i F e n ' + PhNMez . n n m I PhNMep O=FeN P -

PhNMe, +

The NADPH-dependent deoxygenation of N,N-dialkylanilines N-oxides to N,N-dialkylanilines has been previously reported (Sugiura et al., 1976; Iwasaki et al., 1977). This reaction could be demonstrated with some of the N,N-dialkylaniline N-oxides considered here (Table 11). The rate of NADPH-dependent N-dealkylation exceeds that of N,N-dialkylaniline formation except in the case of the 4-cyano derivative. Both of the NADPH-dependent reactions involve Polonovski chemistry (Oae et al., 1963; Craig et al., 1961; Ferris et al., 1968) (Scheme 7). Presumably the NADPH- independent reaction involves Fe(II1) and the NADPH-dependent reaction involves Fe(I1). Polonovski reactions occur with ferrous iron and in acid (Oae et al., 1963; Craig et al., 1961; Ferris et al., 1968), and a limited amount of this chemistry may be available to P450s. I t is certainly not dominant (e.g. compare rates of N-dealkylation of N,N-dialkylanilines and their N-oxides (Table 11)) but it is more rapid than the N-dealkylation reactions observed in the absence of NADPH. We have not tried to correct the rates of N-oxygenation (Table 111) for the rates of N-oxide reduction, as we feel that they do not make a substantial contribution and do not affect the conclusions reached about the process of N-oxygenation in this study. Furthermore, the addition of unlabeled N,N-dimethylaniline N-oxide to an incubation did not affect the level of radiolabeled N-oxide formed from N,N-dimethyl[ ring- "Claniline.

The formation of N,N-dimethylaniline from N,N-dimethylaniline N-oxide could not be demonstrated in the work of Heimbrook et al. (1984) with Pseudomonas putida P450 101 or in our own studies with rat P450 2B1 (see above). Further- more, N,N-dimethylaniline N-oxides have not been shown to support the oxidation of other P450 substrates (i.e. camphor by P450 101 (Heimbrook et al., 1984) or aminopyrine by P450

2B1 (Burka et al., 1985)). In the present study, we found that P450 2B1 would not catalyze the formation of I4C-labeled N- methylaniline when mixed with N,N-dimethyl[ring-'4C]aniline and N,N-dimethylaniline N-oxide. These results can be contrasted with the case of iodosylbenzene-supported N,N- dimethylaniline N-demethylation, which is catalyzed by P450 2B1 at rates as high as 400 nmol min" (nmol of P450)" (Macdonald et al., 1989). The high rate of the iodosylbenzene- supported reaction argues that either (i) the rate of exchange of substrate and product in the P450 2B1 active site must be very rapid (i.e. >400 min"), or (ii) that there is room for both iodosylbenzene and N,N-dimethylaniline to occupy the active site at the same time. With regard to the latter possibility, examination of space-filling models suggests that the space occupied by a combination of iodosylbenzene and N,N-dimethylaniline is no larger than that required for some of the larger substrates of P450 2B1. It follows that one possibility would be for N,N-dimethylaniline N-oxide and N,N-dimethylaniline to both be present in the active site. Thus, the suggestion (Heimbrook et al., 1984) that the reaction of a putative Fe03+ species and N,N-dimethylaniline is so rapid as to preclude oxidation in rabbit P450 2B4, an apparent ortholog of rat P450 2B1, appears to be unfounded. Even the bulky 4-tert-butyl-2,6-bis[l-hydroxyl-l-trifluoromethyl)- 2,2,2-trifluoroethyl]iodobenzene is oxidized in an iodosylbenzene-dependent reaction a t a rate of >38 min" (Guengerich, 1989a). A more likely alternative to heterolytic scission is that the cleavage of the N-0 bond of the N,N-dimethylaniline N - oxide is homolytic and only the formal Fe02+ state is then achieved in the P450. As indicated in Scheme 2, the Fe02+/ aminium radical pair would be expected to collapse rapidly with deprotonation of the a-hydrogen and radical recombination to give the (unstable) carbinolamine, as in the normal


SCHEME 7. Postulated mechanism of N-dealkylation of N-oxides pro- ceeding in the presence of ferrous P460.

CH3

reaction. Indeed this pathway is simply a microscopic reversal of what is believed to be the normal pathway of formation of N-oxides by P450s (Scheme 1). Since it is now clear that model metalloporphyrins can be oxidized, albeit slowly, by N,N-dimethylaniline N-oxides with production of N,N-dimethylanilines (Shannon and Bruice, 1981; Nee and Bruice, 1982), the ligands and environment may be critical in govern- ing the nature of the cleavage of the N-oxides. There is also ample precedent for both homolytic and heterolytic cleavage of hydroperoxides by metalloporphyrins and hemoproteins under different conditions (Labeque and Marnett, 1989).

Acknowledgments-We thank Professors T. L. Macdonald and L. J. Marnett for comments on the manuscript, M. V. Martin for the preparation of P450 2B1, C. G . Turvy and M. V. Martin for preparation of NADPH-P450 reductase, and J. Palmer for assistance in preparation of the manuscript.

REFERENCES

Alterman, M. A., and Dowgii, A. I. (1990) Biomed. Chromatogr. 4, 221-222 Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Bid. Chem.

Baba, T., Yamada, H., Oguri, K., and Yoshimura, H. (1987) Xenobiotica 17,

Bondon, A,, Macdonald, T. L., Harris, T. M., and Guengerich, F. P. (1989) J.

Bowry, V. W., and Ingold, K. U. (1991) J. Am. Chem. SOC. 113,5699-5707 Brodie, B. B., Gillette, J. R., and LaDu, B. M. (1958) Annu. Rev. Biochem. 27,

Burka, L. T., Guengerich, F. P., Willard, R. J., and Macdonald, T. L. (1985) J.

Burstyn, J. N., Iskandar, M., Brady, J. F., Fukuto, J. M., and Cho, A. K. (1991)

Caahman, J. R. (1989) Mol. Pharmacol. 36,497-503 Craig, J. C., and Purushothaman, K. K. (1970) J. Org. Chem. 35,1721-1722 Craig, J. C., Dwyer, F. P., Glazer, A. N., and Horning, E. C. (1961) J. Am.

Damani, L. A,, Patterson, L. H., and Gorrod, J. W. (1978) J. Chromatogr. 155,

267,11288-11295

1029-1038

Biol. Chem. 264,1988-1997

427-454

Am. Chem. SOC. 107, 2549-2551

Chem. Res. Toxrcol. 4, 70-76

Chem. SOC. 83,1871-1878

227-2AQ Dinnocenzo, J. P., and Banach, T. E. (1989) J. Am. Chem. SOC. 111, 8646-

Ferris, J. P., Genve, R. D., and Gapski, G. R. (1968) J. Org. Chem. 33, 3493-

Frederick, C. B., Mays, J. B., Ziegler, D. M., Guengerich, F. P., and Kadlubar,

Fu'imori, K., Takata, T., Fujiware, S., Kikuchi, O., and Oae, S. (1986) Tetra-

Gorrod, J. W. (1981) Eur. J. Drug Metab. Pharmacokinet. 6, 195-206 Groves, J. T., and Gilbert, J. A. (1986) Inorg. Chem. 26, 123-125

Guengerich, F. P. (1984) J. Med. Chem. 27, 1101-1103 Guengerich, F. P. (1978) Biochemistry 17,3633-3639

Guengerich, F. P. (1989a) J. Biol. Chem. 264, 17198-17205 Guengerich, F. P. (1989b) in Principles and Methods of Toxicology (Hayes, A.

". ""

8653

3498

F. F. (1982) Cancer Res. 42,2671-2677

kedron Lett. 27,1617-1620

W., ed) pp. 777-814, Raven Press, New York

RPh--r;l: + O=Fe' RPh-N : HQFe" I I

I

CH2 CH20H II I I I

RPh-N + RPh-N : Feu

CH3

RPhNHCH3 + HCHO

Guengerich, F. P. (1990) Crit. Reu. Biochem. Mol. Biol. 26,97-153 Guengerich, F. P., and Bijcker, R. H. (1988) J. Biol. Chem. 263,8168-8175 Guengerich, F. P., and Macdonald, T. L. (1984) Acc. Chem. Res. 17, 9-16 Guengerich, F. P., and Macdonald, T. L. (1990) FASEB J. 4,2453-2459 Guengerich, F. P., and Martin, M. V. (1980) Arch. Biochem. Biophys. 205,

Guengerich, F. P., and Strickland, T. W. (1977) Mol. Pharmacol. 13,993-1004 Guengerich, F. P., Muller-Enoch, D., and Blair, I. A. (1986) Mol. Pharmacol.

365-379

3n. 287-205 Guengerich, F. P., Willard, R. J., Shea, J. P., Richards, L. E., and Macdonald,

- - , - - . - - -

T. L. (1984) J. Am. Chem. SOC. 106. 6446-6447 Hammons, G. J., Guengerich, F. P., Weis, C. C.: Beland, F. A,, and Kadlubar,

Hayashi, Y., and Yamazaki, 1. (1979) J. Biol. Chem. 254,9101-9106 Heimbrook, D. C., Murray, R. I., Egeberg, K. D., Sligar, S. G., Nee, M. W., and

Hlavica, P., and Hulsmann, S. (1979) Biochem. J . 182,109-116 Iwasaki, K., Noguchi, H., Kato, R., Imai, Y., and Sato, R. (1977) Biochem.

Kurebayashi, H. (1989) Arch. Biochem. Biophys. 270,320-329 Labeque, R., and Marnett, L. J. (1989) J. Am. Chem. Soc. 111,6621-6627 La:bmet-Johnson, P. R., and Ziegler, D. M. (1986) J. Biochem. Toxicol. 1, 15-

F. F. (1985) Cancer Res. 46,3578-3585

Bruice, T. C. (1984) J. Am. Chem. SOC. 106,1514-1515

Biophys. Res. Commun. 77, 1143-1149

Lee, W. A., Caldenvood, T. S., and Bruice, T. C. (1985) Proc. Natl. Acad. Sci. L1

U. S. A. 82.4301-4305 Macdonald, T. L., Zirvi. K.. Burka. L. T.. Pevman. P.. and Guengerich. F. P.

~~~ ~~ ~

(1982) J. Am. Chem. Soc.'104, 2050-2052 ' '

Biochemistry 28,2071-2077

Y I

Macdonald, T. L., Gutheim, W. G., Martin, R. B., and Guengerich, F. P. (1989)

Mansuy, D., Battioni, P., and Battioni, J. P. (1989) Eur. J. Biochem. 184. 267-

Masui, M., and Sayo, H. (1971) J. Chem. SOC. Vol., 1593-1596 Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol.

Muller-Enoch, D., Churcbill, P., Fleischer, S., and Guengerich, F. P. (19M) J.

Nee, M. W., and Bruice, T. C. (1982) J. Am. Chem. SOC. 104, 6123-6125 Nelsen, S. F., and Ippoliti, J. T. (1986) J. Am. Chem. SOC. 108,4879-4881 Nordblom, G. D., and Coon, M. J. (1977) Arch. Biochem. Biophys. 180, 343-

Oae, S., Kitao, T., and Kawamura, S. (1963) Tetrahedron 19, 1783-1788 Okazaki, O., and Guengerich, F. P. (1993) J. Biol. Chem. 268,1546-1552 Omura, T., and Sato, R. (1964) J. Biol. Chem. 239,2370-2378 Ortiz de Montellano, P. R., and Stearns, R. A. (1987) J. Am. Chem. SOC. 109,

Ostovic, D., Knobler, C. B., and Bruice, T. C. (1987) J. Am. Chem. Soc. 109,

Palmer, G., and Reedijk, J. (1992) J. Biol. Chem. 267,665-677 Pandey, R. N., Armstrong, A. P., and Hollenberg, P. F. (1989) Biochem.

PIe, P., and Marnett, L. J. (1989) J. Bid. Chem. 264, 13983-13993 Prough, R. A., Brown, M. I., Dannan, G. A., and Guengerich, F. P. (1984)

Shannon, P., and Bruice, T. C. (1981) J. Am. Chem. SOC. 103,4580-4582 Shono, T. (1984) Tetrahedron 40,811-850 Smith, P. J., and Mann, C. K. (1969) J. Urg. Chem. 34, 1821-1826 Sugiura, M., Iwasaki, K., and Kato, R. (1976) Mol. Pharmacol. 12,322-334 Thurman, R. G., Ley, H. G., and Scholz, R. (1972) Eur. J. Biochem. 25, 420-

Traher, W., and Karrer, P. (1958) Helu. Chim. Acta 41,2066-2074 Walsh, C. (1979) Enzymatic Reaction Mechanisms, W. H. Freeman Co., San

285

Chem. 2.58, 14445-14449

Bid. Chem. 269,8174-8182

347

3415-3420

3444-3451

Pharmacol. 38, 2181-2185

Cancer Res. 44,543-548

430

Francisco

N-Oxide Formation and Decomposition by P450 9997 Watanabe, Y., Iyanagi, T., and Oae, S. (1980) Tetrahedron Lett. 21,3685-3688 C. (1989) Drug Metab. Dispos. 17,387-392 Watanabe, Y., Numata, T., Iyanagi, T., and Oae, S. (1981) Bull. Chem. SOC. Wittig, G., and Sommer, H. (1955) J. Liebig’s Ann. Chem. 594, 1-14

Watanabe, Y., Iyanagi, T., and Oae, S. (1982a) Tetrahedron Lett. 23,533-536 (1979) Full. Chern. SOC. Jpn. 52,3208-3212 Watanabe, Y., Oae, S., and Iyanagi, T. (1982b) Bull. Chm. SOC. Jpn. 55,188- Yasukochl, Y., and Masters, B. S . S. (1976) J. BioL Chem. 251,5337-5344

Weinberg, N. L., and Brown, E. A. (1966) J. Org. Chem. 31,4058-4061 Ziegler, D. M. (1991) Drug Metab. Dispos. 19, 847-852 Ziegler, D. M., and Pettit, F. H. (1964) Biochem. Biophys. Res. Cornrnun. 15,

Williams, D. E., Reed, R. L., Kedzierski, B., Guengerich, F. P., and Buhler, D. 188-193

Jpn. 64, 1163-1170 Yasukochi, K., Taniguchi, I., Yamaguchi, H., Miyaguchi, K., and Horie, K.

195

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, · PDF fileis the dominant P450 reaction...

Documents

Transcript of THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, · PDF fileis the dominant P450 reaction...