Synthesis of Stereospecifically Deuterated 4-(Methylnitrosamino)-1-(3

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Chem. Res. Toxicol. 2003, 16, 782-793

Synthesis of Stereospecifically Deuterated 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) Diastereomers and Metabolism by A/J Mouse Lung Microsomes and Cytochrome P450 2A5 John R. Jalas†,‡ and Stephen S. Hecht*,‡ Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, and Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455 Received February 10, 2003

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in mice and rats and is a putative human lung carcinogen. NNK undergoes cytochrome P450-mediated metabolic activation to DNA-binding intermediates but is also extensively reduced to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in vivo. Because NNAL is also tumorigenic, the carcinogenicity of NNK may actually be governed by the metabolic activation of NNAL, rather than direct activation of NNK. Metabolism of NNK and NNAL at the 4-position generates the same critical DNA lesion, O6-methylguanine, the levels of which are correlated to tumorigenicity in the A/J mouse model. In an effort to better understand the bioactivation of NNAL and the effect of carbinol-carbon stereochemistry on prochiral selectivity at the 4-position, (R)- and (S)-NNAL, along with the stereospecifically 4-deuterated diastereomers (1R,4R)-[4-2H1]NNAL, (1R,4S)-[4-2H1]NNAL, (1S,4R)-[4-2H1]NNAL, and (1S,4S)-[4-2H1]NNAL, were synthesized. The in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda-expressed mouse cytochrome P450 2A5. Carbinol-carbon stereochemistry did not appreciably influence stereoselectivity at the 4-position in the metabolism of these compounds by mouse lung microsomes or P450 2A5 but did influence the regiochemistry of metabolism. The ratio of 4- to N-methyl hydroxylation was approximately 1:1 for the A/J mouse lung microsome-mediated metabolism of all substrates, but this ratio was higher for (1S) substrates than for their (1R) counterparts when P450 2A5 was used. Interestingly, P450 2A5 converted substrates with (1S) stereochemistry to the respective N-oxides, but this metabolite was not formed from substrates with (1R) stereochemistry. Furthermore, P450 2A5 catalyzed the formation of NNK from (1S) substrates at significantly greater maximal rates than from (1R) substrates. The implications of these differences in metabolism for the tumorigenic mechanism of NNAL are discussed.

Introduction 1

The nicotine-derived nitrosamine NNK is a component of tobacco products and cigarette smoke, a pulmonary carcinogen in mice and rats, and a putative human lung carcinogen (2-4). NNK undergoes a variety of metabolic transformations, and its ultimate carcinogenicity is intimately tied to its metabolic fate (2). NNK is metabolically activated to electrophilic intermediates that bind DNA, but it is also rapidly and extensively converted to its carbonyl reduction product, NNAL, in vivo and in vitro * To whom correspondence should be addressed. Tel: 612.624.7604. Fax: 612.626.5135. E-mail: [email protected]. † Department of Chemistry, University of Minnesota. ‡ Cancer Center, University of Minnesota. 1 Abbreviations: COSY, 1H-1H correlation spectroscopy; diol, 1-(3pyridyl)-1,4-butanediol; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; KIE, kinetic isotope effect; lactol, 2-hydroxy-5-(3-pyridyl)tetrahydrofuran; LOD, limit of detection; (R)-(-)-MTPA-Cl, (R)-(-)-R-methoxy-R-trifluoromethylphenylacetic acid chloride; NNAL, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol; NNAL-N-oxide, 4-(methylnitrosamino)-1-(3pyridyl-N-oxide)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone; O6-mG, O6-methylguanine; OPB, 4-oxo-4-(3-pyridyl)butanal; OR, NADPH:cytochrome P450 oxidoreductase; pyridyl-THF, 2-(3pyridyl)tetrahydrofuran; Sf9, Spodoptera frugiperda; TEA, thermal energy analyzer (a nitrosamine selective detector).

(2, 5-7). NNAL is also a lung carcinogen in mice and rats, and it is unclear if the carcinogenicity of NNK is due to metabolic activation of NNK itself or to the metabolic activation of NNAL generated from NNK in vivo. The metabolic activation of NNAL is likely mediated by members of the cytochrome P450 superfamily of monooxygenases and consists of hydroxylation of the carbon atoms adjacent to the nitroso group (i.e., R-hydroxylation) (2). Hydroxylation at the R-methylene carbon of NNAL generates the unstable metabolite 2, which spontaneously decomposes to 5 and methanediazohydroxide (4) (Scheme 1). Hydroxy aldehyde 5 cyclizes to lactol. Methanediazohydroxide (4), or the methyldiazonium ion (7), reacts with DNA bases to form methyl adducts such as O6-mG. Hydroxylation of the R-methyl carbon of NNAL forms 3; this unstable R-hydroxynitrosamine spontaneously decomposes to formaldehyde and, ultimately, 8, which reacts with DNA bases to form pyridylhydroxybutyl adducts (8) or with water to form diol. Alternatively, 8 can cyclize to pyridyl-THF (Scheme 1) (2). NNAL is also converted to the noncarcinogenic metabolite NNAL-N-oxide and oxidized to NNK (Scheme 1) (2, 9).

10.1021/tx034021t CCC: $25.00 © 2003 American Chemical Society Published on Web 05/20/2003

Metabolism of Deuterated NNALs

Chem. Res. Toxicol., Vol. 16, No. 6, 2003 783

Scheme 1. Relevant Metabolic Transformations of NNK and NNAL

Scheme 2. Structures of Deuterated NNAL Diastereomers

* Location of 3H in radiolabeled analogues.

In NNK-treated A/J mice, levels of O6-mG in lung DNA correlate strongly with lung tumor multiplicity (10, 11), implying that the R-methylene hydroxylation pathway is crucial to the tumorigenic mechanism. Importantly, 7 is generated from the R-methylene hydroxylation of both NNK and NNAL (Scheme 1), making the role of each of these carcinogens in the overall tumor initiation mechanism unclear. Several lines of evidence indicate that the tumorigenicity of NNK may be mediated by the (S) enantiomer of NNAL in the A/J mouse (Scheme 2) (9, 12). (S)-NNAL and NNK exhibit nearly identical tumorigenicities in the A/J mouse lung, whereas (()NNAL and (R)-NNAL are much less tumorigenic (9).

Moreover, (S)-NNAL is the predominant enantiomer of NNAL generated from NNK in a variety of in vitro systems (12), and it undergoes R-methylene hydroxylation at a faster rate than does (R)-NNAL in A/J mouse lung microsomes (9). Furthermore, two stereospecifically deuterated isotopomers of NNK, (4R)-[4-2H1]NNK and (4S)-[4-2H1]NNK, exhibit differential tumorigenicity in the A/J mouse lung but do not exhibit differential metabolism in A/J mouse lung microsomes (13). (4R)-[42H ]NNK is 2-fold less tumorigenic than its enantiomer, 1 but (4R)-[4-2H1]NNK and (4S)-[4-2H1]NNK exhibit similar KIEs on the formation of the 4-hydroxylation product when they are incubated with A/J mouse lung microsomes (13). Thus, the difference in tumorigenicity between these deuterated NNK isotopomers may be due to differences in metabolism of the corresponding deuterated NNAL diastereomers after the compounds are stereoselectively reduced in vivo; this is depicted in Figure 1. In light of these data, it is hypothesized that (S) stereochemistry at the carbinol carbon dictates alignment of the substrate in the P450 active site such that the pro-R R-methylene hydrogen atom is preferentially abstracted (Figure 1). To probe the effect of carbinol-carbon stereochemistry on P450-mediated 4-hydroxylation of NNAL, (R)- and (S)NNAL, along with the stereospecifically 4-deuterated isotopomers (1R,4R)-[4-2H1]NNAL, (1R,4S)-[4-2H1]NNAL, (1S,4R)-[4-2H1]NNAL, and (1S,4S)-[4-2H1]NNAL (and the respective analogues radiolabeled at the 5-position of the pyridine ring), were synthesized (Scheme 2). The metabolism of these compounds was investigated with A/J mouse lung microsomes and, given the importance of

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Figure 1. Summary of the hypothesis tested in this paper. Given that NNK is rapidly and stereospecifically reduced to NNAL (2, 12), administration of NNK, (4R)-[4-2H1]NNK, and (4S)-[4-2H1]NNK to A/J mice should lead to the presence of (S)NNAL, (1S,4R)-[4-2H1]NNAL, and (1S,4S)-[4-2H1]NNAL in the mouse lung, respectively. Because (S)-NNAL is more tumorigenic and a better R-methylene hydroxylation substrate than its enantiomer (9) and (4R)-[4-2H1]NNK is less tumorigenic than (4S)-[4-2H1]NNK in the A/J mouse lung (13), one would expect to observe the greatest KIE on lactol formation when (1S,4R)[4-2H1]NNAL is a microsomal substrate.

members of the P450 2A subfamily to the metabolism of NNK (13-16), with Sf9-expressed mouse cytochrome P450 2A5.

Experimental Procedures Caution: NNK and NNAL cause cancer in laboratory animals; they are to be handled with extreme care. Appropriate protective clothing and ventilation are to be used at all times. Equipment. Low-resolution mass spectra were obtained on a Finnigan LCQ Deca instrument operated in positive-ion mode (Finnigan MAT/Thermoquest, San Jose, CA). High-resolution FAB-MS was performed by the Department of Chemistry Mass Spectrometry facility (University of Minnesota, Minneapolis, MN). NMR spectra were acquired on a Varian Inova 800 MHz instrument (Varian, Inc., Palo Alto, CA). Enantioselective GC analyses were performed as described (17, 18). Optical rotations were determined using a Jasco DIP-370 digital polarimeter (Jasco, Inc., Easton, MD). UV spectrophotometry was performed on a Beckman DU-7400 instrument (Beckman, Fullerton, CA). Chemicals. NNK, NNAL metabolite standards, (4R)-[4-2H1]NNK, and (4S)-[4-2H1]NNK were synthesized as described (1925). [5-3H]NNK (14 Ci mmol-1, 98% radiochemical purity) was purchased from Moravek Biochemicals, Inc. (Brea, CA) and purified to >99% radiochemical purity by reverse phase HPLC. [5-3H]-(4R)-[4-2H1]NNK (14.9 Ci mmol-1, >99% radiochemical purity) and [5-3H]-(4S)-[4-2H1]NNK (14.9 Ci mmol-1, >99% radiochemical purity) were synthesized by Moravek Biochemicals, Inc. from the appropriate stereospecifically deuterated 5-brominated precursors (13). NADP+, glucose-6-phosphate,

Jalas and Hecht glucose-6-phosphate dehydrogenase, EDTA, barium hydroxide, and zinc sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). Human OR (EC 1.6.2.4) expressed in Sf9 cells was purchased from PanVera (Madison, WI). (R)-(-)-MTPA-Cl was procured from Aldrich Chemical (Milwaukee, WI). Monoflow 5 was purchased from National Diagnostics, Inc. (Atlanta, GA). All other chemicals were of the highest grade commercially available. Synthesis. 1. (R)-NNAL and (S)-NNAL (Scheme 2). After NNK (211 mg, 1.0 mmol) was reduced to (()-NNAL with NaBH4 (22), 198 mg (0.95 mmol) was reacted with (R)-(-)-MTPA-Cl (1.3 g, 5.1 mmol) in CH2Cl2 (26). (R)-NNAL-(S)-MTPA and (S)NNAL-(S)-MTPA (1H NMR, 800 MHz, CDCl3; see Table 1) were separated via normal phase HPLC, and the alcohols were released by base hydrolysis (0.1 N NaOH, 37 °C) and purified by RP-HPLC as described (26). (R)-NNAL (9.3 mg, 8.8%): 1H NMR (800 MHz, CDCl3; see Table 2). ESI-MS m/z (% relative intensity): 210.1 (100, [M + H]+), 180.1 (15). ESI-MS/MS m/z (% relative intensity): 210.0 (55, [M + H]+), 180.0 (100), 162.0 (37). FAB-HRMS (m/z): [M + H]+ calcd for C10H16N3O2, 210.1243; found, 210.1249. Chiral-GC-TEA analysis: >99% R (no S detected); [R]25D +31.3 ( 0.2° (c 0.69, MeOH). (S)-NNAL (12 mg, 11%): 1H NMR (800 MHz, CDCl3; see Table 2). ESIMS m/z (% relative intensity): 210.1 (100, [M + H]+), 180.1 (14). ESI-MS/MS m/z (% relative intensity): 210.0 (59, [M + H]+), 180.0 (100), 162.0 (34). FAB-HRMS (m/z): [M + H]+ calcd for C10H16N3O2, 210.1243; found, 210.1246. Chiral-GC-TEA analysis: >99% S (no R detected); [R]25D -29.2 ( 0.3° (c 1.1, MeOH). 2. (1R,4R)-[4-2H1]NNAL and (1S,4R)-[4-2H1]NNAL (Scheme 2). (4R)-[4-2H1]NNK (112 mg, 0.54 mmol) was converted to (()(4R)-[4-2H1]NNAL and resolved as described above. (1R,4R)-[42H ]NNAL (8.2 mg, 15%): 1H NMR (800 MHz, CDCl ; see Table 1 3 2). ESI-MS m/z (% relative intensity): 211.1 (100, [M + H]+), 210.1 (2), 181.1 (8). ESI-MS/MS m/z (% relative intensity): 211.0 (61, [M + H]+), 181.0 (100), 163.0 (37). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1309; 98.2% 2H. Chiral-GC-TEA analysis: >99% 1R (no 1S detected); [R]25 D +28.2 ( 0.2° (c 0.92, MeOH). (1S,4R)-[4-2H1]NNAL (10.4 mg, 19%): 1H NMR (800 MHz, CDCl3; see Table 2). ESI-MS m/z (% relative intensity): 211.1 (100, [M + H]+), 210.1 (1), 181.2 (8). ESI-MS/MS m/z (% relative intensity): 211.0 (59, [M + H]+), 181.0 (100), 163.0 (35). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1308; 99.3% 2H. Chiral-GCTEA analysis: 98.6% 1S (1.4% 1R); [R]25D -22.2 ( 0.2° (c 0.78, MeOH). 3. (()-(4R)-[4-2H1]NNAL (Scheme 2). (()-(4R)-[4-2H1]NNAL was synthesized from (4R)-[4-2H1]NNK as described (22). 1H NMR (800 MHz, CDCl3; see Table 2). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1309; 99.5% 2H. 4. (1R,4S)-[4-2H1]NNAL and (1S,4S)-[4-2H1]NNAL (Scheme 2). (4S)-[4-2H1]NNK (112 mg, 0.54 mmol) was converted to (()(4S)-[4-2H1]NNAL and resolved as described above. (1R,4S)-[42H ]NNAL (14.1 mg, 25%): 1H NMR (800 MHz, CDCl ; see Table 1 3 2). ESI-MS m/z (% relative intensity): 211.2 (100, [M + H]+), 210.2 (3), 181.2 (8). ESI-MS/MS m/z (% relative intensity): 211.0 (57, [M + H]+), 181.0 (100), 163.0 (34). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1308; 97.0% 2H. Chiral-GC-TEA analysis: >99% 1R (no 1S detected); [R]25 D +31.4 ( 0.3° (c 0.73, MeOH). (1S,4S)-[4-2H1]NNAL (15.6 mg, 28%): 1H NMR (800 MHz, CDCl3; see Table 2). ESI-MS m/z (% relative intensity): 211.2 (100, [M + H]+), 210.2 (4), 181.2 (14). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1309; 96.1% 2H. Chiral-GC-TEA analysis: >99% 1S (no 1R detected); [R]25D -28.7 ( 0.2° (c 1.2, MeOH). 5. (()-(4S)-[4-2H1]NNAL (Scheme 2). (()-(4S)-[4-2H1]NNAL was synthesized from (4S)-[4-2H1]NNK (22). 1H NMR (800 MHz, CDCl3; see Table 2). FAB-HRMS (m/z): [M + H]+ calcd for C10H15DN3O2, 211.1304; found, 211.1309; 98.9% 2H. 6. Radiolabeled Analogues. [5-3H]Labeled analogues were synthesized as described above (scaled down 40-fold), with the following radiolabeled precursors as starting material: [5-3H]-

Metabolism of Deuterated NNALs Table 1.

1H

Chem. Res. Toxicol., Vol. 16, No. 6, 2003 785 NMR (800 MHz) Chemical Shift (δ) Data for NNAL-MTPA Estersa 4-CH (D)

pyridine ring compound

2′

4′

5′

CHOR 6′

E

Z

E a

2-CH2 -CH3

Z b

a

b

E

Z

E a

3-CH2 Z

b

a

E b

(R)-NNAL-(S)-MTPA 8.57 7.50 7.25 8.52 5.94 5.91 4.13 3.65 3.52 2.95 3.68 2.02 1.86 1.93 b (1R,4R)-[4-2H1]NNAL- 8.56 7.48 7.23 8.51 5.94 5.90 4.09 3.40 2.95 3.67 2.02 1.85 1.94 b (S)-MTPA 2 (1R,4S)-[4- H1]NNAL- 8.57 7.49 7.25 8.52 5.94 5.90 4.12 3.62 2.95 3.67 2.02 1.85 1.93 b (S)-MTPA

a

Z b

a

b

1.81 1.72 1.57 1.48 1.81 1.71 1.57 1.48 1.80 1.71 1.56 1.47

(S)-NNAL-(S)-MTPA 8.61 7.63 7.31 8.61 5.99 5.95 4.09 4.05 3.62 3.43 2.89 3.64 1.98 1.80 1.89 b 1.67 1.63 (1S,4R)-[4-2H1]NNAL- 8.59 7.61 7.29 8.59 5.99 5.95 4.08 3.59 2.90 3.63 1.98 1.79 1.89 1.74 1.66 1.62 (S)-MTPA (1S,4S)-[4-2H1]NNAL- 8.59 7.62 7.30 8.59 5.99 5.95 4.02 c 2.89 3.62 1.97 1.80 1.88 b 1.66 1.61 (S)-MTPA

1.41 1.40 1.40

a Compounds were synthesized as described in the Experimental Procedures. Samples were dissolved in CDCl . In the (1R) series, the 3 chemical shifts of the MTPA phenyl protons were at δ7.34 (3H) and δ7.38 (2H), and the methoxy protons were at δ3.48. In the (1S) series, the chemical shifts of the MTPA phenyl protons were at δ7.34 (2H) and δ7.38 (3H), and the methoxy protons were at δ3.42. b Resonance obscured by E-3a or E-3b. c Resonance obscured by -OCH3.

Table 2.

1H

NMR (800 MHz) Chemical Shift (δ) Data for Stereospecifically Deuterated NNAL Diastereomersa 4-CH2 (D) pyridine ring

compound

2′

4′

5′

CHOH 6′

E

Z

E a

3-CH2 -CH3

Z b

a

b

E

Z

E a

2-CH2 Z

bb

ab

E ab

Z bc

ac

b

-OH

(()-NNAL 8.46 7.68 7.26 8.42 4.76 4.72 4.15 3.65 3.59 3.00 3.71 1.91 1.79 1.66 1.79 1.70 1.70 1.59 2.10 (()-(4R)-[4-2H1]NNAL 8.60 7.73 7.34 8.54 4.82 4.80 4.18 4.14 3.68 3.58 3.01 3.73 1.91 1.81 d 1.81 1.73 1.73 d e (()-(4S)-[4-2H1]NNAL 8.59 7.70 7.33 8.54 4.81 4.78 4.17 4.13 3.65 3.58 3.00 3.72 1.92 1.81 d 1.81 1.72 1.72 d e (R)-NNAL 8.52 7.70 7.29 8.46 4.79 4.75 4.17 3.66 3.59 3.00 3.72 1.92 1.81 1.67 1.81 1.71 1.71 1.60 2.94 (1R,4R)-[4-2H1]NNAL 8.51 7.67 7.28 8.47 4.78 4.74 4.13 3.57 3.00 3.72 1.91 1.80 1.66 1.80 1.72 1.72 1.58 3.22 (1R,4S)-[4-2H1]NNAL 8.49 7.69 7.27 8.44 4.78 4.73 4.16 3.63 3.00 3.71 1.90 1.80 1.66 1.80 1.70 1.70 1.60 3.17 (S)-NNAL 8.50 7.69 7.28 8.45 4.78 4.74 4.16 3.65 3.59 3.00 3.72 1.90 1.80 1.65 1.80 1.72 1.72 1.59 3.05 (1S,4R)-[4-2H1]NNAL 8.52 7.68 7.28 8.48 4.79 4.74 4.17 3.64 3.01 3.72 1.92 1.81 1.68 1.81 1.73 1.73 1.59 3.07 (1S,4S)-[4-2H1]NNAL 8.48 7.69 7.27 8.43 4.78 4.73 4.12 3.55 2.99 3.71 1.90 1.79 1.65 1.79 1.70 1.70 1.58 3.19 a Compounds were synthesized as described in the Experimental Procedures. Samples were dissolved in CDCl . b Overlapping resonances, 3 (E)-3-CH2b and (E)-2-CH2a. c Overlapping resonances, (E)-2-CH2b and (Z)-2-CH2a. d Resonance obscured by impurity. e -OH proton not observed.

2NNK for (()-[5-3H]NNAL, (R)-[5-3H]NNAL, and (S)-[5-3H]NNAL; [5-3H]-(4R)-[4-2H1]NNK for (()-[5-3H]-(4R)-[4-2H1]NNAL, [5-3H]-(1R,4R)-[4-2H1]NNAL, and [5-3H]-(1S,4R)-[4-2H1]NNAL; and [5-3H]-(4S)-[4-2H1]NNK for (()-[5-3H]-(4S)-[4-2H1]NNAL, [5-3H]-(1R,4S)-[4-2H1]NNAL, and [5-3H]-(1S,4S)-[4-2H1]NNAL. The radiolabeled starting materials were diluted to 4 mCi µmol-1 with the appropriate nonradiolabeled ketone prior to the NaBH4 reduction. It was important to carry out the esterification reaction in a minimal volume of CH2Cl2 (150 µL) to achieve acceptable yields (≈75%). All radiolabeled compounds were purified by RP-HPLC (radiochemical purity >99%). Typically, 500 µCi of each pure alcohol was obtained from 8 mCi of starting ketone (12.5% radiochemical yield). Molar Absorptivity of NNAL. The molar absorptivity of (()-NNAL was determined with weighed samples (9-11 mg). UV (95% EtOH) λmax, nm (): 230 (7990 ( 268 M-1 cm-1). In Vitro Metabolism: Mouse Lung Microsomes. Female A/J mice, 5-6 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME), housed in groups of five under standard conditions (27), and maintained on AIN-93G diet (Dyets, Inc., Bethlehem, PA) for 1 week. Mouse lung microsomes were prepared (28) and stored at -80 °C until use. Protein was quantified using the Sigma Microprotein kit (Sigma Chemical Co.) with bovine serum albumin as the standard. Incubations were performed essentially as described (29), except a glucose6-phosphate concentration of 25 mM was used, 1 mM EDTA was added, and substrate concentrations were (1 µCi unless otherwise indicated) 0.25 (0.3 µCi), 0.50 (0.6 µCi), 0.75 (0.9 µCi), 1.0, 2.5, 5.0, 10, 15, 25, or 50 µM, in a total volume of 200 µL. Reactions were initiated by the addition of the NADPHgenerating system and incubated at 37 °C for 45 min. Reactions were quenched, filtered, and analyzed as described (29), with slight modifications (13) and with Monoflow 5 as the scintillant

(pumped at 4 mL min-1). Quantitation was achieved by measuring the radioactivity that coeluted with unlabeled standards (LOD for all metabolites: =0.10 pmol). Kinetic analyses were performed in quadruplicate (four data points per substrate concentration). In Vitro Metabolism: Mouse Cytochrome P450 2A5. Microsomes prepared from Sf9 cells transfected with the cDNA of P450 2A5 (4.5 pmol P450 mL-1) were a generous gift from Dr. Sharon Murphy, University of Minnesota (14). P450 2A5 (1.5 pmol) was preincubated with a 25-fold molar excess of OR for 5 min at 37 °C to facilitate association of these membrane preparations before the reaction components were added. Omission of this preincubation step resulted in an approximately 5-fold reduction in maximal activity. Substrate concentrations were the same as those used with mouse lung microsomes. Incubations (30 min) and analyses were carried out as described (13, 29), with the exceptions mentioned above for the mouse lung microsome experiment. Kinetic assays with P450 2A5 were done in quadruplicate (four data points per substrate concentration). Data Analysis. Kinetic parameters were determined using the EZ-Fit software package (Perrella Scientific, Amherst, NY), which fits the data to the Michaelis-Menten equation using nonlinear least-squares regression (30). Statistical analyses (Student’s t-test) were performed with SigmaStat (Jandel Scientific, San Rafael, CA); a P value < 0.05 was deemed significant.

Results Synthesis and Characterization of Stereospecifically Deuterated NNAL Diastereomers. Stereospecifically 4-deuterated NNAL diastereomers and their

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respective radiolabeled analogues were synthesized via resolution of the corresponding MTPA-esters (26). After reaction with (R)-(-)-MTPA-Cl and normal phase HPLC separation, 1H NMR spectra of the NNAL-MTPA esters were obtained and assignments were made based on previous results (26) and according to the convention of Ohtani et al. (31) (see Table 1). NNAL exists as an equilibrium mixture of interconverting E and Z rotamers (Scheme 2), both of which can be observed by 1H NMR (Tables 1 and 2) (32). When the carbinol carbon has the (R) configuration, the phenyl ring of the MTPA ester deshields the pyridine ring 2′- and 6′-protons to a similar extent, such that they can be clearly resolved (as they are in the free alcohols) (Tables 1 and 2). Conversely, the 2′- and 6′-protons of the (1S) diastereomeric esters have equivalent chemical shifts (Table 1). In this case, the phenyl ring has a greater deshielding effect on the pyridyl 6′-proton than the 2′-proton. The effect of the MTPA group on all chemical shift values was consistent with the convention of Ohtani et al. (31), and these chemical shift values were consistent with the proposed structures (Scheme 2). 1H-1H COSY experiments confirmed all assignments. The desired alcohols were obtained via base hydrolysis of the respective esters; 1H NMR chemical shift assignments for the resolved alcohols, as well as the respective racemic alcohols, are presented in Table 2. The R-methylene protons of (()-NNAL are diastereotopic and are a complex multiplet in the E configuration (δ 4.15, Table 2, Figure 2A) but have nonequivalent chemical shifts in the Z configuration (δ 3.65 and 3.59, Table 2, Figure 2A). The R-methylene proton of (()-(4R)-[4-2H1]NNAL exhibits two distinct chemical shifts in both the E (δ 4.18, 4.14) and the Z rotamers (δ 3.68, 3.58), respectively (Table 2, Figure 2B). Two signals for each rotamer were observed because the sample contains an equal mixture of (1R) and (1S) isomers. The same phenomenon was observed with (()-(4S)-[4-2H1]NNAL (Table 2, Figure 2C). The R-methylene proton of E-(1R,4S)[4-2H1]NNAL was deshielded slightly relative to the R-methylene proton of E-(1R,4R)-[4-2H1]NNAL (δ 4.16 vs 4.13, Table 2, Figure 2E,F), indicating that the R-methylene proton is being deshielded by the hydroxyl group when both groups are on the same “side” of the molecule (of course, this is an average of many conformations). The deshielding effect was more pronounced in the Z rotamers (δ 3.63 vs 3.57, Table 2, Figure 2E,F), probably because the slight deshielding effect of the hydroxyl group is accentuated when the R-methylene proton is being shielded by the nitroso moiety (32).2 The deshielding effect of the hydroxyl group on the 4-proton was also observed in the E- and Z-(1S) diastereomers, consistent with the proposed assignments (Table 2, Figure 2H,I). The chemical shift assignments in Table 2 were consistent with the assigned structures; 1H-1H COSY experiments confirmed all assignments. Chiral-GC-TEA, optical rotation, and mass spectral data were also consistent with the proposed structures (see the Experimental Procedures and Supporting Information). In Vitro Metabolism: Mouse Lung Microsomes. Deuterated NNAL diastereomers were converted to lactol, diol, and NNAL-N-oxide by A/J mouse lung microsomes (Tables 3 and 4). A normal KIE on the apparent 2 The diamagnetic anisotropy of a nitroso group shields protons that are in the Z configuration relative to the nitroso oxygen, whereas it deshields protons that are in the E configuration (32). Thus, the 4-protons of E-NNAL are downfield from the 4-protons in Z-NNAL.

Jalas and Hecht

Figure 2. Partial 800 MHz 1H NMR spectra of deuterated NNAL diastereomers (CDCl3, δ 3.5-4.3). (A) (()-NNAL, (B) (()(4R)-[4-2H1]NNAL, (C) (()-(4S)-[4-2H1]NNAL, (D) (R)-NNAL, (E) (1R,4R)-[4-2H1]NNAL, (F) (1R,4S)-[4-2H1]NNAL, (G) (S)-NNAL, (H) (1S,4R)-[4-2H1]NNAL, and (I) (1S,4S)-[4-2H1]NNAL.

Vmax (DV)3 of lactol formation was only observed for the (()-(4S)-[4-2H1]NNAL substrate (DV ) 1.2 ( 0.1, Table 3), while inverse KIEs of 0.67-0.93 were seen for all other deuterated substrates (Table 3). The Vmax/Km values for lactol formation among the nine substrates were quite similar (1.1 ( 0.2 to 1.9 ( 0.5), even though the Vmax values ranged from 5.6 ( 0.4 ((()-(4S)-[4-2H1]NNAL) to 21 ( 0.9 pmol mg-1 min-1 ((1S,4S)-[4-2H1]NNAL) (Table 3). The apparent Vmax and Km values for diol formation were similar to the respective values for lactol formation 3 The convention of Northrop (1) is followed for the designation of kinetic deuterium isotope effects: DV ) HVmax / DVmax and D(V/K) ) H(V D max/Km)/ (Vmax /Km).



Table 3. Kinetic Parameters for r-Hydroxylation of Deuterated NNAL Diastereomers by A/J Mouse Lung Microsomesa metabolites and kinetic parameters substrate

Vmaxb

Km (µM)

Vmax/Km

DV

D(V/K)

(()-NNAL (()-(4R)-[4-2H1]NNAL (()-(4S)-[4-2H1]NNAL

6.6 ( 0.3 7.2 ( 0.4c 5.6 ( 0.4

5.7 ( 0.9 4.5 ( 0.8 3.0 ( 0.7

1.2 ( 0.2 1.6 ( 0.3 1.9 ( 0.5

0.92 ( 0.07 1.2 ( 0.1

0.72 ( 0.18 0.62 ( 0.18

(R)-NNAL (1R,4R)-[4-2H1]NNAL (1R,4S)-[4-2H1]NNAL

6.5 ( 0.4 8.7 ( 0.5d 7.2 ( 0.4

5.7 ( 1 5.6 ( 1 5.8 ( 1

1.1 ( 0.2 1.6 ( 0.3 1.2 ( 0.2

0.75 ( 0.06 0.90 ( 0.08

0.73 ( 0.19 0.92 ( 0.25

(S)-NNAL (1S,4R)-[4-2H1]NNAL (1S,4S)-[4-2H1]NNAL

14 ( 0.8e 15 ( 0.7f 21 ( 0.9g

9.9 ( 1.5 8.6 ( 1.2 17 ( 1.6g

1.4 ( 0.2 1.7 ( 0.3 1.2 ( 0.1

0.93 ( 0.07 0.67 ( 0.05

0.81 ( 0.18 1.1 ( 0.2

lactol

diol (()-NNAL (()-(4R)-[4-2H1]NNAL (()-(4S)-[4-2H1]NNAL

6.0 ( 0.3 6.0 ( 0.3 6.0 ( 0.3

3.7 ( 0.7 3.0 ( 0.6 3.1 ( 0.6

1.6 ( 0.3 2.0 ( 0.4 1.9 ( 0.4

1.0 ( 0.1 1.0 ( 0.1

0.81 ( 0.23 0.84 ( 0.23


5.8 ( 0.3 8.1 ( 0.5d 6.7 ( 0.3

5.0 ( 0.9 5.0 ( 1f 4.1 ( 0.6

1.2 ( 0.2 1.6 ( 0.3 1.6 ( 0.3

0.72 ( 0.06 0.87 ( 0.06

0.72 ( 0.20 0.71 ( 0.17


15 ( 0.6e 18 ( 0.6f 26 ( 1g

7.8 ( 1 8.9 ( 0.8 18 ( 1.6g

1.9 ( 0.3 2.0 ( 0.2 1.4 ( 0.1

0.83 ( 0.04 0.58 ( 0.03

0.95 ( 0.16 1.3 ( 0.2

a Kinetic parameters were determined over the substrate concentration range of 0.25-50 µM as described in the Experimental Procedures. Values are means ( SE from four replicates (four data points per substrate concentration). b Units are pmol mg-1 min-1. c Value is significantly different from the corresponding value for (()-(4S)-[4-2H1]NNAL, P < 0.05. d Value is significantly different from the corresponding value for (R)-NNAL, P < 0.05. e Value is significantly different from the corresponding value for the other two undeuterated substrates, P < 0.05. f Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 4-position, P < 0.05. g Value is significantly different from the corresponding value for substrates with the same stereochemistry at either the 1- or the 4-position, P < 0.05.

Table 4. Kinetic Parameters for N-Oxidation of Deuterated NNAL Diastereomers by A/J Mouse Lung Microsomesa kinetic parameters for NNAL-N-oxide substrate

Vmaxb

Km (µM)

Vmax/Km

DV

D(V/K)

(()-NNAL (()-(4R)-[4-2H1]NNAL (()-(4S)-[4-2H1]NNAL

0.4c

20 ( 22 ( 0.7 20 ( 0. 7

2.2 ( 0.2 2.1 ( 0.3 2.1 ( 0.3

9.1 ( 0.8 10 ( 2 9.5 ( 1.4

0.91 ( 0.03 1.0 ( 0.04

0.87 ( 0.15 0.96 ( 0.17


13 ( 0.4d 16 ( 0.6 18 ( 0.6

2.0 ( 0.2 1.4 ( 0.2 1.5 ( 0.2

6.5 ( 0.7 11 ( 1.7 12 ( 1.6

0.81 ( 0.04 0.72 ( 0.03

0.57 ( 0.10 0.54 ( 0.09

9.4 ( 0.5c 8.8 ( 0.4e 21 ( 1f

11 ( 0.6 12 ( 0.6 8.5 ( 0.5

1.0 ( 0.03 0.58 ( 0.02

0.94 ( 0.07 1.3 ( 0.1


103 ( 2c 103 ( 2e 178 ( 5f

a Kinetic parameters were determined over the substrate concentration range of 0.25-50 µM as described in the Experimental Procedures. Values are means ( SE from four replicates (four data points per substrate concentration). b Units are pmol mg-1 min-1. c Value is significantly different from the corresponding value for the other two undeuterated substrates, P < 0.05. d Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 1-position, P < 0.05. e Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 4-position, P < 0.05. f Value is significantly different from the corresponding value for substrates with the same stereochemistry at either the 1- or the 4-position, P < 0.05.

for each substrate; these two metabolites were formed in an approximately 1:1 ratio (Table 3). NNAL-N-oxide was the major metabolite formed upon incubation of all nine substrates with A/J mouse lung microsomes (Table 4). Substrates with (1S) stereochemistry were converted to their N-oxides with apparent Vmax values that were about an order of magnitude higher than when racemic or (1R) substrates were used, but the Km values were also significantly higher, leading to roughly equivalent Vmax/ Km values (Table 4). In general, the Km values were higher for all metabolites when substrates with (1S) stereochemistry were utilized than with (() or (1R) substrates (Tables 3 and 4). The Km values for all products from (1S,4S)-[4-2H1]NNAL were uniformly higher than those for the other eight substrates (Tables 3 and 4). NNK was also formed from all substrates, but saturating conditions were not reached in this experi-

ment. The rates of NNK formation were about 5 pmol mg-1 min-1 for all substrates at substrate concentrations of 50 µM (data not shown). Pyridyl-THF was not observed under these conditions. The rates of metabolite formation increased linearly over a microsomal protein concentration range of 0.10-0.75 mg mL-1 and incubation times up to 60 min. No metabolites were detected when the NADPH-generating system was omitted or when heattreated microsomes were used. In Vitro Metabolism: Sf9-Expressed Mouse Cytochrome P450 2A5. P450 2A5 catalyzed the oxidation of NNAL enantiomers and deuterated diastereomers to lactol, diol, pyridyl-THF, NNAL-N-oxide, and NNK (Tables 5-7). A statistically significant reduction in Vmax for lactol formation was observed when (1R,4R)-[4-2H1]NNAL was a P450 2A5 substrate (DV ) 1.4 ( 0.1, Table 5). Slight KIEs on lactol formation were also seen when (1S,4R)-

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Jalas and Hecht

Table 5. Kinetic Parameters for r-Methylene Hydroxylation of Deuterated NNAL Diastereomers by Sf9-Expressed Mouse Cytochrome P450 2A5a kinetic parameters for lactol Vmaxb

Km (µM)

Vmax/Km

(()-NNAL

0.55 ( 0.02c

1.7 ( 0.3

0.32 ( 0.06


0.42 ( 0.02 0.30 ( 0.02d 0.44 ( 0.04

2.1 ( 0.5 2.5 ( 0.6 2.9 ( 0.9


0.45 ( 0.03 0.38 ( 0.03 0.35 ( 0.02e

0.78 ( 0.26 1.6 ( 0.6 1.5 ( 0.3

substrate

DV

D(V/K)

0.20 ( 0.05 0.12 ( 0.03 0.15 ( 0.05

1.4 ( 0.1 0.96 ( 0.10

1.7 ( 0.6 1.3 ( 0.5

0.58 ( 0.22 0.24 ( 0.09 0.23 ( 0.05

1.2 ( 0.1 1.3 ( 0.1

2.4 ( 1.3 2.5 ( 1.1

a Kinetic parameters were determined over the substrate concentration range of 0.25-50 µM as described in the Experimental Procedures. Values are means ( SE from four replicates (four data points per substrate concentration). b Units are pmol min-1 pmol P450-1. c Value is significantly different from the corresponding value for the other two undeuterated substrates, P < 0.05. d Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 1-position, P < 0.05. e Value is significantly different from the corresponding value for (S)-NNAL, P < 0.05.

Table 6. Kinetic Parameters for r-Methyl Hydroxylation of Deuterated NNAL Diastereomers by Sf9-Expressed Mouse Cytochrome P450 2A5a metabolites and kinetic parameters DV

D(V/K)

0.11 ( 0.008 0.091 ( 0.006 0.11 ( 0.01

1.2 ( 0.05 0.92 ( 0.04

1.2 ( 0.1 1.0 ( 0.1

0.13 ( 0.04 0.075 ( 0.01 0.062 ( 0.01

0.42 ( 0.03 0.47 ( 0.04

1.8 ( 0.6 2.1 ( 0.8

0.027 ( 0.007 0.027 ( 0.005 0.028 ( 0.009

0.80 ( 0.12 0.87 ( 0.18

1.0 ( 0.3 0.97 ( 0.40

0.012 ( 0.008 0.017 ( 0.007 0.024 ( 0.005

0.79 ( 0.28 1.4 ( 0.44

0.69 ( 0.53 0.50 ( 0.34

Vmaxb

Km (µM)

0.47 ( 0.01c

5.1 ( 0.5c

0.092 ( 0.009


0.03c

1.2 ( 1.0 ( 0.03d 1.3 ( 0.05e

11 ( 11 ( 0.7e 12 ( 1e


0.17 ( 0.01f 0.41 ( 0.02 0.36 ( 0.02

1.3 ( 0.4f 5.5 ( 0.7 5.8 ( 1

(()-NNAL

0.77 ( 0.10

46 ( 9.7

0.017 ( 0.004


0.96 ( 0.11 1.2 ( 0.1e 1.1 ( 0.2e

35 ( 7.2 44 ( 6.6e 39 ( 11e


0.19 ( 0.06c 0.24 ( 0.04 0.14 ( 0.01

16 ( 9.2 14 ( 5.0 5.9 ( 1.1

substrate

Vmax/Km diol

(()-NNAL

0.8c

pyridyl-THF

a Kinetic parameters were determined over the substrate concentration range of 0.25-50 µM as described in the Experimental Procedures. Values are means ( SE from four replicates (four data points per substrate concentration). b Units are pmol min-1 pmol P450-1. c Value is significantly different from the corresponding value for the other two undeuterated substrates, P < 0.05. d Value is significantly different from the corresponding value for substrates with the same stereochemistry at either the 1- or the 4-position, P < 0.05. e Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 4-position, P < 0.05. f Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 1-position, P < 0.05.

[4-2H1]NNAL and (1S,4S)-[4-2H1]NNAL were substrates (DV ) 1.2 ( 0.1 and 1.3 ( 0.1, respectively, Table 5). Substrates with (1R) stereochemistry exhibited higher maximal rates for the formation of R-methyl hydroxylation metabolites (diol and pyridyl-THF) than the R-methylene hydroxylation product (lactol), whereas the opposite trend was observed with (S)-NNAL (Tables 5 and 6). (1S,4R)-[4-2H1]NNAL and (1S,4S)-[4-2H1]NNAL were metabolized to diol and lactol at similar maximal rates (Tables 5 and 6). Diol or pyridyl-THF was the major metabolite for (1R) substrates (e.g., diol Vmax ) 1.2 ( 0.03 pmol min-1 pmol-1 for (R)-NNAL, Table 6), whereas NNK was the major metabolite for (1S) substrates (e.g., Vmax ) 1.8 ( 0.05 pmol min-1 pmol-1 for (1S,4R)-[4-2H1]NNAL, Table 7). However, lactol was formed with the greatest efficiency from all seven substrates (see Vmax/Km values, Tables 5-7). NNAL-N-oxide was formed when (()-NNAL or (1S) substrates were incubated with P450 2A5 but not from (1R) substrates (Table 7). Substrates with (1S) stereochemistry were converted to NNK with greater efficiency (Vmax/Km ) 0.13-0.19, Table 7) than (1R)

substrates (Vmax/Km ) 0.017-0.019, Table 7). The Vmax/ Km value for NNK formation from (()-NNAL was 0.056 ( 0.008, intermediate between those for the (1R) and (1S) compounds (Table 7). Rates of metabolite formation were linear with respect to time at incubation times up to 30 min (except for pyridyl-THF, the rate of which was only linear up to incubation times of 10 min) and over a P450 2A5 concentration range of 1.25-15 nM (data not shown). No metabolites were detected when the NADPH-generating system was omitted, when heat-treated microsomes were used, or when microsomes prepared from nontransfected Sf9 cells were used. NNAL was not a substrate for mouse P450 2A4, which differs in primary sequence from P450 2A5 by only 11 amino acids (out of 494) (33).

Discussion Stereospecifically 4-deuterated NNAL diastereomers were synthesized and characterized, and their kinetic parameters were determined using A/J mouse lung microsomes and Sf9-expressed mouse cytochrome P450 2A5.



Table 7. Kinetic Parameters for Metabolism of Deuterated NNAL Diastereomers by Sf9-Expressed Mouse Cytochrome P450 2A5: NNAL-N-oxide and NNK Formationa metabolites and kinetic parameters substrate

Vmaxb

Km (µM)

Vmax/Km

DV

D(V/K)

0.46 ( 0.04 0.55 ( 0.04

1.7 ( 0.7 2.2 ( 0.9

NNAL-N-oxide (()-NNAL (R)-NNAL (1R,4R)-[4-2H1]NNAL (1R,4S)-[4-2H1]NNAL

0.17 ( 0.01

4.7 ( 1.1c

0.036 ( 0.01

0.15 ( 0.05 0.083 ( 0.01 0.067 ( 0.01

ND d ND ND


0.16 ( 0.01e 0.35 ( 0.02e 0.29 ( 0.01e

1.1 ( 0.4e 4.2 ( 0.7 4.3 ( 0.6

(()-NNAL

0.78 ( 0.04

14 ( 1.8

0.056 ( 0.008


0.31 ( 0.43 ( 0.04g 0.33 ( 0.06g

16 ( 3.9 23 ( 3.9 19 ( 8

0.019 ( 0.005 0.019 ( 0.004 0.017 ( 0.008

0.72 ( 0.10 0.94 ( 0.19

1.0 ( 0.3 1.1 ( 0.6


0.78 ( 0.03e 1.8 ( 0.05e 1.3 ( 0.04e

4.2 ( 0.6e, f 13 ( 0.8e 9.7 ( 0.9

0.19 ( 0.03 0.14 ( 0.01 0.13 ( 0.01

0.43 ( 0.02 0.60 ( 0.03

1.3 ( 0.2 1.4 ( 0.2

NNK 0.03f

a Kinetic parameters were determined over the substrate concentration range of 0.25-50 µM as described in the Experimental Procedures. Values are means ( SE from four replicates (four data points per substrate concentration). b Units are pmol min-1 pmol P450-1. c Value is significantly different from the corresponding value for (S)-NNAL, P < 0.05. d Not detected. e Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 1-position, P < 0.05. f Value is significantly different from the corresponding value for the other two undeuterated substrates, P < 0.05. g Value is significantly different from the corresponding value for substrates with the same stereochemistry at the 4-position, P < 0.05.

Although the hypothesized effects of carbinol-carbon stereochemistry on the stereoselectivity of R-methylene hydroxylation were not observed (see Figure 1), incubation of these substrates with both in vitro systems resulted in some interesting differences in regioselectivity and kinetics that may have a bearing on the tumorigenic mechanism of NNAL. When stereospecifically 4-deuterated NNAL diastereomers were incubated with A/J mouse lung microsomes, substrates with (1S) stereochemistry were converted to lactol, diol, and NNAL-N-oxide at higher maximal rates than were substrates with (1R) stereochemistry or the respective racemates (Tables 3 and 4). The Km values were also higher for the (1S) substrates, however; thus, the Vmax/Km values were comparable among all substrates for any given metabolite and were lower than the values observed for NNK metabolism by A/J mouse lung microsomes (Figure 3A) (13, 16). The Vmax data were in concordance with an earlier study that demonstrated greater metabolic activation of (S)-NNAL than (R)-NNAL in A/J mouse lung microsomes, although that study utilized only one substrate concentration (1 µM) (9). The similarities in Vmax/Km values for R-hydroxylation of both (R)- and (S)-NNAL by A/J mouse lung microsomes cloud the hypothesis that differential metabolic activation of NNAL enantiomers is responsible for their differing tumorigenicities (9, 12); this will be discussed below. When Sf9-expressed P450 2A5 was used as the catalyst, there were some pronounced effects of carbinolcarbon stereochemistry on the regiochemistry of metabolism. Substrates with (1S) stereochemistry were preferentially converted to NNK, presumably via hydroxylation of the 1-carbon and subsequent dehydration of a gemdiol intermediate (Tables 5-7) (34, 35). The (1R) substrates, on the other hand, were preferentially metabolized at the N-methyl position. The Vmax values for the formation of diol and pyridyl-THF were similar among the three (1R) substrates, although the Km values were much higher for pyridyl-THF formation (Table 6). Pyr-

Figure 3. Comparison of Vmax/Km values for (A) female A/J mouse lung microsome or (B) Sf9-expressed mouse cytochrome P450 2A5-mediated metabolism of NNK, (()-NNAL, (R)-NNAL, and (S)-NNAL. Data for NNK are from ref 13. Metabolism assays were conducted as described in the Experimental Procedures. All assays were done over the substrate concentration range of 0.25-50 µM. Vmax/Km values for pyridyl-THF were omitted from B.

idyl-THF and diol are formed from a common precursor (6 or 8, Scheme 1); thus, the rate of their formation is likely affected by the stability of this intermediate under these experimental conditions. The Km values for pyridylTHF formation may not reflect true Michaelis-Menten kinetic parameters because they probably consist of both chemical and enzymatic rate constants. Further evidence for this comes from the fact that the rate of diol formation

790


was linear with respect to incubation time (up to 30 min, the time used for the kinetic assays), whereas the rate of pyridyl-THF formation was independent of incubation time after 10 min, implying that its rate of formation is not entirely enzyme-dependent (data not shown, see Results). Regardless, both metabolites were derived from R-methyl hydroxylation of NNAL and were clearly in greatest abundance when (1R) compounds were used as substrates. The most dramatic effect of carbinol-carbon stereochemistry on P450 2A5-mediated metabolism of NNAL isotopomers was on oxidation of the pyridine nitrogen. Racemic NNAL and the (1S) substrates were converted to their respective N-oxides at modest maximal rates, but no N-oxidation was seen with any of the (1R) substrates (Table 7). Considering that N-oxidation of NNAL is a detoxification pathway (9), it was surprising that the more tumorigenic enantiomer was detoxified to a much greater extent by this P450 (although enhanced Noxidation of (S)-NNAL relative to (R)-NNAL was observed in this and a previous study using A/J mouse lung microsomes (9)). Given that N-oxides are the major products of NNK and NNAL metabolism by mouse lung microsomes (Figure 3A), the relatively low rates of NNAL N-oxidation by P450 2A5 observed in this study and the lack of NNK-N-oxide formation by this P450 observed previously (Figure 3B) (13, 14) indicate that P450 2A5 does not play a major role in the microsomal N-oxidation of these nitrosamines in the mouse lung. Previous work has demonstrated that the metabolism of NNK by cytochrome P450 2A5 results in the formation of only the two R-hydroxylation products (13, 14), whereas the NNAL diastereomers investigated here were converted to their R-hydroxylation products, N-oxides, and NNK. Moreover, NNK was metabolized with greater efficiency than was (()-NNAL or its individual enantiomers by this P450, as can be seen in Figure 3B. Upon investigation of Figure 3B, it appears that (S)-NNAL is metabolized at the R-methylene position with greater efficiency than is (R)-NNAL and with comparable efficiency to NNK, but these results should be interpreted with caution due to the relatively high standard error in the Km values (Table 5 and Figure 3B). What is clear, however, is that NNK is a better substrate for both A/J mouse lung microsomes and P450 2A5 than is NNAL (Figure 3A,B). Considering that P450s possess a relatively hydrophobic active site and generally catalyze the metabolism of lipophilic substrates, the decreased lipophilicity of NNAL relative to NNK may be responsible for the decreased rates of P450-mediated metabolism; these lower rates may indicate that metabolic activation of NNAL is not as critical for tumorigenesis as is metabolic activation of NNK. As mentioned previously, (S)-NNAL is significantly more tumorigenic than (()- or (R)-NNAL in the A/J mouse lung and is as tumorigenic as NNK (9). It was proposed that the enhanced tumorigenicity of (S)-NNAL relative to (R)-NNAL in A/J mice was due to greater metabolic activation of the former compound and greater glucuronidation of the latter (9, 12). In this study, (S)NNAL was metabolically activated at the R-methylene position by mouse lung microsomes at a 2.5-fold greater rate than was (R)-NNAL (14 ( 0.8 vs 6.5 ( 0.4 pmol mg-1 min-1, Table 3) but was metabolically detoxified (NNALN-oxide formation) at an 8-fold greater rate (103 ( 2 vs 13 ( 0.4 pmol mg-1 min-1, Table 4). Moreover, even

Jalas and Hecht

though the Vmax values were greater for (S)-NNAL than (R)-NNAL, the Vmax/Km values for lactol and diol formation from (R)- and (S)-NNAL were similar. Thus, the differential tumorigenicity of (R)- and (S)-NNAL may not be due to differential metabolic activation of these two carcinogens. Recent data on (R)- and (S)-NNAL pharmacokinetics in the rat suggest that the enhanced tumorigenicity of (S)-NNAL may be due to its preferential retention in the lungspossibly by binding to a β-adrenergic receptor (36). Furthermore, in the urine of humans that have quit using tobacco products, the (S):(R)-NNAL ratio increases over time, again suggesting preferential retention of (S)-NNAL (or enhanced excretion of (R)NNAL) (37). Thus, pharmacokinetic effects may be more important than metabolic activation for the tumorigenicity of (S)-NNAL. It would be interesting to determine if A/J mouse lung DNA O6-mG levels differ over time after the administration of (R)- or (S)-NNAL. If the metabolic activation of (S)-NNAL is not a primary factor in its tumorigenic mechanism, then perhaps conversion to NNK and subsequent metabolism of the ketone are more important. Isotope effects on the conversion of NNAL to NNK have been studied in vivo, but there were no differences in tumorigenicity between NNAL and [1-2H1]NNAL in the A/J mouse lung (38). The lack of an isotope effect implies that the conversion of NNAL to NNK is not a rate-limiting step in the tumorigenic mechanism; however, racemic material was utilized in that study (38). Considering that (S)-NNAL was converted to NNK at a faster rate than was (R)-NNAL by P450 2A5 (Table 7) and by rat and mouse lung microsomes (9, 12), it is possible that the tumorigenicity of (S)-NNAL is mediated by enhanced conversion to NNK; this hypothesis could be tested by evaluating the tumorigenicity of (S)-[1-2H1]NNAL and (R)-[1-2H1]NNAL in the A/J mouse. Also, comparing the rates of (S)- and (R)-NNAL oxidation to NNK by dehydrogenases, which are more likely to be responsible for this reaction in vivo than is a P450, should shed light on this topic. The importance of the conversion of NNAL to NNK in the tumorigenic mechanism is further illustrated by the observation that administration of NNAL to rats leads to the detection of HPB-releasing DNA and Hb adducts, as opposed to the diol-releasing adducts that would be expected from the direct metabolic activation of NNAL (39). Thus, it is possible that the tumorigenic mechanism of NNK in the A/J mouse lung is highly dependent on the selective formation and retention of (S)-NNAL and its subsequent reconversion to NNK, the preferred substrate for metabolic activation; this is summarized in Figure 4. Furthermore, it appears that the greater tumorigenicity of (S)-NNAL relative to its enantiomer is not mediated by direct hydroxylation, but rather by enhanced conversion to NNK, although other mechanisms are certainly possible (Figure 4). Deuterium substitution did not result in any statistically significant normal KIEs on lactol formation when A/J mouse lung microsomes were employed as the catalyst. Instead, inverse KIEs were observed for lactol formation with some of the substrates (DV ) 0.75 ( 0.06 for (1R,4R)-[4-2H1]NNAL and 0.67 ( 0.05 for (1S,4S)-[42 H1]NNAL, Table 3). It is possible that deuterium substitution at one prochiral position leads to enhanced abstraction of the remaining proton and thus an observed inverse KIE. This explanation is unlikely to account for the effects seen here, however, because inverse KIEs were



Figure 4. Summary of NNK and NNAL transformations in the A/J mouse based on results in this paper and previous results (9, 12, 13). Bold arrows depict pathways thought to be important in tumorigenesis, while arrow length depicts the relative extents to which the glucuronidation, N-oxidation, and NNK formation pathways occur when comparing (R)- and (S)-NNAL.

observed independent of substrate stereochemistry at C-4 (Table 3). If the hypothesis that (1S) stereochemistry enhances the abstraction of the pro-R R-methylene hydrogen atom was correct, then one would have expected to see the greatest KIE on lactol formation when (1S,4R)-[4-2H1]NNAL was the substrate (see Figure 1), but there was no difference between the Vmax values for lactol formation from (S)-NNAL and (1S,4R)-[4-2H1]NNAL (DV ) 0.93 ( 0.07, Table 3). It is possible that intrinsic isotope effects were masked, but these substrates are not amenable to the competitive intramolecular experimental design used to unmask the intrinsic isotope effect (40, 41). However, because multiple products are formed from a single substrate, the effect of deuterium substitution on branching among the various metabolic pathways can be used to estimate the intrinsic isotope effect (41). From the kinetic analyses (Tables 3-7), it can be seen that deuterium substitution did not markedly affect the conversion of the various substrates to different metabolites; thus, it was decided to forego the isotopically sensitive branching approach and instead seek information about the stereospecificity of the R-methylene hydroxylation of NNAL by analyzing directly for the deuterium content of lactol. Unfortunately, despite extensive efforts, reliable and reproducible MS detection of the lactol derived from mouse lung microsomes or P450 2A5 could not be achieved. Preliminary LC/MS data (not shown) demonstrated, however, that the deuterium content of the lactol formed upon incubation of these substrates with A/J mouse lung microsomes or P450 2A5 was the same (≈80%), irrespective of the stereochemistry (at C1 or C4) of the substrate. The absence of stereoselectivity is consistent with the concept that the substrate has considerable mobility in the active site such that the active oxygen species can select protium over deuterium; this is not an uncommon occurrence in P450-mediated reactions (42, 43). The modest isotope effects observed with P450 2A5 could be partially due to the contribution of an R-2° KIE. If the first-formed R-hydroxynitrosamine (2, Scheme 1) possesses a deuterium atom, then a small, normal R-2° KIE upon decomposition to 5 (Scheme 1) of 1.1-1.4 would be expected because the hybridization changes from sp3 to sp2 in the transition state (44).

Interestingly, P450 2A5 catalyzed the R-methylene hydroxylation of (4R)-[4-2H1]NNK with a pronounced KIE (DV ) 2.2 ( 0.2), but a KIE was not observed when the substrate was (4S)-[4-2H1]NNK (DV ) 1.1 ( 0.1) (13). That a substantial KIE was not observed with any of the NNAL substrates (Table 5) implies that the ketone moiety is more important than the hydroxyl group in aligning the substrate in the P450 active site such that pro-4R stereoselectivity is effected. Although the specific hypothesis tested in this works that (S)-carbinol-carbon stereochemistry promotes pro-R selectivity at the R-methylene position of NNALswas not borne out, the stereospecifically deuterated NNAL diastereomers synthesized here may serve as useful probes of P450-substrate interactions. Several noteworthy differences in kinetic parameters were observed among the (R)- and (S)-NNAL enantiomers that may have a bearing on the carcinogenicity of these compounds, as discussed above. Currently, a molecular model of cytochrome P450 2A5 is being constructed based on the crystal structure of rabbit P450 2C5 (45). Docking (R)- or (S)-NNAL in the active site of this model should aid in explaining the effects of carbinol-carbon stereochemistry on the regioselectivity of P450 2A5-mediated metabolism. Furthermore, the increased mobility of NNAL in the active site of P450 2A5 relative to NNK may provide important clues to critical enzyme-substrate interactions in the active site. Detailed molecular information regarding specific P450-substrate interactions that lead to metabolic activation of tobacco-specific carcinogens should aid in the search for agents that prevent tobacco-related cancers.

Acknowledgment. This work was supported by a National Cancer Institute Grant CA-81301. S.S.H. is an American Cancer Society Research Professor, supported by Grant RP-00-138. We thank Dr. Sharon Murphy for the generous gift of cytochrome P450s 2A4 and 2A5 and Dr. Mark Distefano for helpful discussions. We are also thankful for the kind provision of the stereospecifically deuterated NNK starting materials by Dr. Edward McIntee and for NNK and NNAL metabolite standards by Dr. Pramod Upadhyaya. Expert assistance with

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HPLC, MS, and NMR was provided by Steven Carmella, Dr. Pete Villalta, and Dr. Beverly Ostrowski, respectively, and is gratefully acknowledged. We appreciate the assistance of Ky-Anh Le with the chiral-GC analyses and the aid of Patrick Kenney with animal maintenance and tissue harvesting. The expertise of Bob Carlson during manuscript preparation is gratefully acknowledged. NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation. Supporting Information Available: 1H NMR and MS spectra of (R)-NNAL-(S)-MTPA, (S)-NNAL-(S)-MTPA, (1R,4R)[4-2H1]NNAL-(S)-MTPA, (1R,4S)-[4-2H1]NNAL-(S)-MTPA, (1S, 4R)-[4-2H1]NNAL-(S)-MTPA, (1S,4S)-[4-2H1]NNAL-(S)-MTPA, (R)-NNAL, (S)-NNAL, (1R,4R)-[4-2H1]NNAL, (1R,4S)-[4-2H1]NNAL, (1S,4R)-[4-2H1]NNAL, and (1S,4S)-[4-2H1]NNAL. This material is available free of charge via the Internet at http:// pubs.acs.org.

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TX034021T

Synthesis of Stereospecifically Deuterated 4-(Methylnitrosamino)-1-(3

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