THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 1985 by The American Society of Biological Chemists, Inc.
`
`Vol. 260, No.6, Issue of March 25, pp. 3330-3336, 1985
`Printed in U.S.A.
`
`Branchpoint for Heme Alkylation and Metabolite Formation in the
`Oxidation of Arylacetylenes by Cytochrome P-450*
`
`(Received for publication, August 13, 1984)
`
`Paul R. Ortiz de Montellano and Elizabeth A. Komives
`From the Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center, University of California,
`San Francisco, California 94143
`
`Phenylaeetylene and biphenylaeetylene are oxidized
`by cytochrome P-450 to the corresponding arylacetic
`acids. The acetylenic hydrogen shifts to the adjacent
`carbon and one atom of molecular oxygen is incorpo(cid:173)
`rated into the carboxylic acid group in these transfor(cid:173)
`mations, which are subject to a large kinetic isotope
`effect when the acetylenic hydrogen is replaced by
`deuterium. The same products and isotope effects are
`observed when the two arylacetylenes are oxidized by
`m-chloroperbenzoic acid rather than by the enzyme.
`In contrast, the inactivation of cytochrome P-450 that
`occurs during the oxidation of phenylacetylene is in(cid:173)
`sensitive to deuterium substitution. The partition ratio
`between metabolite formation and enzyme inactivation
`consequently changes from 26 to 15 in going from
`phenylacetylene to the deuterated analogue. Metabo(cid:173)
`lite formation therefore diverges from heme alkylation
`very early in the catalytic process.
`
`The epoxidation of olefins by cytochrome P-450 could
`involve simultaneous formation of bonds between the acti·
`vated oxygen and the two carbons of the 1r-bond, or formation
`of the two bonds in discrete steps separated by an ionic or
`radical intermediate. Retention of the olefin stereochemistry
`in the epoxides favors a concerted epoxidation mechanism
`(1-4), but the fact that the prosthetic heme of cytochrome P-
`450 is alkylated during the oxidation of terminal olefins favors
`a nonconcerted mechanism. Heme1 alkylation, which also
`occurs with retention of the olefin stereochemistry (4), is
`observed when the activated oxygen is transferred to the
`internal carbon of the 1r-bonds (5-9). Additional support for
`a nonconcerted mechanism is provided by the reports that
`1,2-chlorine migration precedes rather than follows epoxide
`formation from halogenated olefins (10, 11), that aldehydes
`and ketones are formed as trace metabolites from certain
`olefins in a process that does not involve the epoxide (11, 12),
`and that the oxidation of styrene is subject to a secondary
`isotope effect when deuterium is located on the internal but
`not the terminal carbon of the 1r-bond (13).
`The cytochrome P-450-catalyzed oxidation of a carbon(cid:173)
`carbon triple bond, a reaction formally related to olefin epox(cid:173)
`idation, has only been unambiguously demonstrated in the
`
`metabolism of biphenylacetylenes (14-19) but is implied by
`the excretion of phenylacetic acid when animals are treated
`with phenylacetylene (20) and by rearrangements observed in
`the metabolism of 17-ethynyl sterols (9). The catalytic oxi(cid:173)
`dation of biphenylacetylene yields biphenylacetic acid in a
`reaction subject to a kinetic isotope effect of 1.4 when the
`acetylenic hydrogen is replaced by deuterium (19). The pos(cid:173)
`sibility that oxygen is inserted into the acetylenic carbon(cid:173)
`hydrogen bond, suggested by the isotope effect, is ruled out
`by quantitative shift of the acetylenic hydrogen to the vicinal
`carbon during the oxidation (17-19) and by the observation
`of a similar intramolecular hydrogen shift in the chemical
`oxidation of biphenylacetylene (17, 18). The enzymatic oxi(cid:173)
`dation of acetylenes, like that of olefins, thus involves reaction
`of the activated oxygen with the 1r-bond. In agreement with
`this, the turnover of acetylenes by cytochrome P-450 results
`in alkylation of the prosthetic heme group (21-23) and the
`formation of heme adducts similar to those obtained with
`terminal olefins (7, 24, 25). We describe here a mechanistic
`investigation of the oxidation of 1r-bonds by cytochrome P-
`450 that exploits the hydrogen shift associated with triple
`bond oxidation as an experimental probe.
`
`MATERIALS AND METHODS2
`
`RESULTS
`Incorporation of Oxygen into the Biphenylacetylene Metab(cid:173)
`olite-Biphenylacetylene was incubated with hepatic micro(cid:173)
`somes from phenobarbital-pretreated rats under an 180 2 at(cid:173)
`mosphere, and the resulting biphenylacetic acid was isolated
`and methylated with diazomethane. Mass spectrometric anal(cid:173)
`ysis (Fig. 1) of the esterified metabolite established that
`approximately 75% of one oxygen in the carboxylic acid
`moiety derived from labeled molecular oxygen. This fractional
`incorporation of label points to catalytic incorporation of one
`atom of molecular oxygen because some dilution of the label
`by oxygen not removed in the purging operations is unavoid(cid:173)
`able. The second oxygen in the carboxyl group, by implication,
`derives from the medium.
`Kinetic Isotope Effect in the Metabolism of Biphenylacety(cid:173)
`lene-The kinetic isotope effect for the oxidation of biphen(cid:173)
`ylacetylene was remeasured to confirm the value reported
`earlier (kH/kD = 1.42) (19). Labeled and unlabeled biphen-
`
`*This research was supported by Grant GM 25515 from the
`National Institutes of Health. Support for facilities used in the
`investigation was provided by Liver Center Core Facility Grant P-30
`AM 26743 and by Grant AM 30297. The costs of publication of this
`article were defrayed in part by the payment of page charges. This
`article must therefore be hereby marked "advertisement" in accord(cid:173)
`ance with 18 U.S.C. Section 1734 solely to indicate this fact.
`1 Heme is used in this paper for iron protoporphyrin IX regardless
`of the oxidation state of the iron or the porphyrin.
`
`2 Portions of this paper (including "Materials and Methods," Ta(cid:173)
`bles 1 and 2, and Figs. 1, 3, 4, and 6) are presented in miniprint at
`the end of this paper. Miniprint is easily read with the aid of a
`standard magnifying glass. Full size photocopies are available from
`the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda,
`MD 20814. Request Document No. 84M-2538, cite the authors, and
`include a check or money order for $4.00 per set of photocopies. Full
`size photocopies are also included in the microfilm edition of the
`Journal that is available from Waverly Press.
`
`3330
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`
`Mechanism of 1r-Bond Oxidation
`
`3331
`
`~~~----~--------------------------,
`
`ylacetylene were incubated with hepatic microsomes from
`phenobarbital-induced rats and aliquots were taken from the
`incubation mixtures at 10-min intervals. Biphenylacetylene
`and biphenylacetic acid were extracted into methylene chlo(cid:173)
`ride, and their concentrations were determined spectroscopi(cid:173)
`cally. This spectroscopic assay is feasible because the absor(cid:173)
`bance maximum of biphenylacetylene is at 273 nm whereas
`that of biphenylacetic acid is at 254 nm, although direct
`spectroscopic analysis of the incubation mixture is not pos(cid:173)
`sible because an interfering chromophore is present in the
`assay mixture that is eliminated in the extraction procedure.
`Metabolite formation is limited to the first 10 min of the
`incubation because the same amount of metabolite is present
`at 30 as at 10 min. The isotope effect calculated from the data
`is kH/ko = 1.38 (Table 1), a value within experimental error
`of that obtained earlier by a gas chromatographic assay (19).
`Isotope Effects on the Destruction of Cytochrome P-450 by
`Arylacetylenes-Biphenylacetylene causes NADPH-
`and
`time-dependent loss of cytochrome P-450 when incubated
`with hepatic microsomes from phenobarbital-pretreated rats
`(Table 2). The enzyme loss determined by spectroscopic quan(cid:173)
`titation of the ferrous-carbon monoxide complex, however,
`does not exceed 4-5% of the total microsomal enzyme. This
`loss occurs within the first 10 min and is not increased if the
`incubation is prolonged a further 20 min. Essentially identical
`results are obtained when the incubations are carried out with
`(1-2H)biphenylacetylene (Table 2). The quantitative reliabil(cid:173)
`ity of these values, however, is compromised by their small
`magnitude and by the fact that they reflect a 4% correction
`for the enzyme lost in the absence of substrates.
`The completion of both metabolite formation and enzyme
`inactivation within the same 10 min period suggests that one
`isozyme, representing no more than 4-5% of the cytochrome
`P-450 in microsomes from phenobarbital-induced rats, is in(cid:173)
`volved in both processes. This result, unexpected in view of
`the report that phenobarbital and 3-methylcholanthrene
`stimulate biphenylacetylene metabolism (14), led us to inves(cid:173)
`tigate whether the cytochrome P-450 loss could be amplified
`by pretreatment with clofibrate, an inducer of cytochrome P-
`450 enzymes involved in fatty acid hydroxylation (27, 28), or
`Arochlor 1254, an inducer of multiple isozymes (29, 30).
`However, microsomal enzyme loss from Arochlor 1254-treated
`rats is the same as that from phenobarbital-treated rats while
`enzyme loss from clofibrate-treated rats is negligible (Table
`2). These results suggest that biphenylacetylene is a specific
`substrate for a minor, phenobarbital-inducible, isozyme.
`Phenylacetylene was found earlier to cause time- and
`NADPH dependent loss of cytochrome P-450 when incubated
`with hepatic microsomes from phenobarbital-pretreated rats
`(22). Losses of 18, 25, and 27% were observed after 10, 20,
`and 30 min of incubation with 10 mM phenylacetylene. A
`hepatic pigment with the absorption spectrum of anN-alkyl(cid:173)
`protoporphyrin IX derivative was furthermore isolated from
`the livers of rats injected with phenylacetylene (22). The
`larger amplitude of the cytochrome P-450 loss and the evi(cid:173)
`dence that phenylacetylene alkylates the prosthetic heme of
`cytochrome P-450 led us to use it as an alternative probe of
`the oxidative mechanism. The rates of inactivation of cyto(cid:173)
`chrome P-450 by phenylacetylene and [1-2H]phenylacetylene
`appear, within experimental error, to be identical (Fig. 2).
`The Metabolism of Phenylacetylene-No information is
`available on the metabolism of phenylacetylene except for the
`report that phenylacetic acid is excreted by rabbits injected
`with phenylacetylene (20). Phenylacetylene therefore was in(cid:173)
`cubated with hepatic microsomes from phenobarbital-pre(cid:173)
`treated rats and the metabolites were isolated by extraction
`
`liOOtJ
`i :l: 40
`
`'f
`a.
`
`¢
`
`0
`
`10
`5
`T•me(min)
`
`15
`
`FIG. 2. The metabolism of phenylacetylene to phenylacetate
`(0) and [l-2H}phenylacetylene to [2-'aH)phenylacetate (D) by
`hepatic microsomes from phenobarbital-induced rats. The loss
`of cytochrome P-450 caused by each of these two compounds (symbols
`as in the main panel) as a semilog function of time, assuming that
`the 30% maximum loss of cytochrome P-450 observed in long term
`incubations is equal to 100% of the vulnerable enzyme, is given in
`the inset.
`
`into diethyl ether. The diethyl ether fraction, after esterifi(cid:173)
`cation with diazomethane, was analyzed by gas chromatog(cid:173)
`raphy. The only quantitatively significant (>5%) metabolite
`detected in the extracts is the methyl ester of phenylacetic
`acid (Fig. 3). A specific search was made for acetophenone,
`but none was detected. The metabolite was identified as
`phenylacetic acid by direct gas chromatographic and mass
`spectrometric comparison with an authentic sample (not
`shown).
`The pattern of metabolites obtained with [1-2H]phenyla(cid:173)
`cetylene is the same as that obtained with the unlabeled
`substrate, but the molecular ion of the methyl phenylacetate
`metabolite is 1 mass unit higher (Fig. 4). The peak for the
`fragment obtained by decarboxylation of the molecular ion
`retains the difference of 1 mass unit. It is thus evident that
`the acetylenic hydrogen of phenylacetylene, like that of bi(cid:173)
`phenylacetylene, shifts quantitatively to the vicinal carbon
`on oxidation of the triple bond.
`Kinetic Isotope Effect in the Metabolism of Phenylacety(cid:173)
`lene-The formation of phenylacetic acid from phenylacety(cid:173)
`lene and [1-2H]phenylacetylene in incubations with hepatic
`microsomes from phenobarbital-pretreated rats was quanti(cid:173)
`tated as a function of time by gas chromatographic analysis
`(Fig. 2). Deuterium substitution significantly retards enzy(cid:173)
`matic oxidation of the triple bond and gives rise to a kinetic
`isotope effect kH/kn
`1.80 (Table 1).
`The isotope effect for phenylacetylene metabolism was
`independently measured by incubating a 1:1 mixture of phen(cid:173)
`ylacetylene and [1-2H]phenylacetylene with hepatic micro(cid:173)
`somes and quantitating the ratio of the deuterated to undeu(cid:173)
`teratedphenylacetic acid metabolites by gas chromatography(cid:173)
`mass spectrometry. The kinetic isotope effect obtained by this
`internal competition method (kH/kn
`1.60) (Table 1) con(cid:173)
`firms that the oxidation ofphenylacetylene by cytochrome P-
`450 is subject to a major isotope effect.
`Isotope Effects on the Chemical Oxidation of Arylacety(cid:173)
`lenes-The oxidation of biphenylacetylene by m-chloroper(cid:173)
`benzoic acid in methylene chloride with a trace of methanol
`yields methyl 2-biphenylacetate as the only detectable prod(cid:173)
`uct. The ratio of the rates of reaction with biphenylacetylene
`and [1-2H)biphenylacetylene reflects an isotope effect kH/kn
`1.38 (Table 1). Attempts to study the oxidation of phen-
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`
`3332
`Mechanism of 1r-Bond Oxidation
`1.5.-------------------.
`
`N
`
`I
`N
`
`N
`I
`N
`
`~ 0.3
`
`2
`Time (hr)
`FIG. 5. The oxidation of phenylacetylene to phenylacetate
`(0) and [l-2H)phenylacetylene to [2- 2H)phenylacetate (0) by
`m-chloroperbenzoic acid in anhydrous benzene.
`
`4
`
`5
`
`ylacetylene under the same conditions, however, were unsuc(cid:173)
`cessful due to the low reactivity of this substrate. The oxida(cid:173)
`tion of phenylacetylene was therefore examined in anhydrous
`benzene in the absence of proton sources as described in an
`earlier study (31). A kinetic isotope effect kH/ko = 1.73 is
`obtained from the rates of product formation from phenyla(cid:173)
`cetylene and [1-2H]phenylacetylene under these conditions
`(Fig. 5). Addition of methanol to the reaction mixture altered
`the kinetics and attenuated the isotope effect, but these
`changes were not investigated.
`
`DISCUSSION
`The oxidation of [1-2H]phenylacetylene by both cyto(cid:173)
`chrome P-450 and m-chloroperbenzoic acid yields phenyla(cid:173)
`cetic acid with the acetylenic deuterium atom shifted to the
`benzylic carbon. The oxidation of phenylacetylene thus pro(cid:173)
`ceeds with a 1,2-hydrogen shift identical to that shown to
`occur previously in the oxidation of biphenylacetylene (17
`18). The oxidation of carbon-carbon triple bonds with con~
`comitant shift of the acetylenic hydrogen thus appears to be
`a general, if frequently only minor, metabolic process.3 The
`analogy between the enzymatic and chemical processes is
`strengthened by the incorporation of a labeled oxygen atom
`into the carboxylic acid function when biphenylacetylene is
`incubated under 180 2 because this labeling pattern is that
`expected if biphenylacetylene is oxidized to biphenylketene
`which then reacts with a molecule of water. The chemical
`oxidation of acetylenes is believed to proceed through such a
`ketene mechanism (32).
`The oxygen atom is bound to the terminal carbon in the
`arylacetylene metabolites but to the internal carbon in the
`heme adducts (Scheme 1) (7, 24, 25). The terminal carbon in
`the heme adducts is the site to which the protoporphyrin IX
`prosthetic group is bound. The reaction regiochemistry ob(cid:173)
`served in the metabolites is favored by rate-determining ox(cid:173)
`ygen addition to the 1r-bond because any electron deficiency
`that develops in the transition state can be stabilized by
`conjugation with the phenyl ring. The absence of acetophe(cid:173)
`none among the metabolites in the chemical and enzymatic
`reactions supports the view that metabolites stem from deliv(cid:173)
`ery of the oxygen to the terminal carbon. The fact that
`
`3 Enzymatic oxidation of the triple bond in an alkyl acetylene ( 4-
`phenylbutyne) with concomitant 1,2-shift of the hydrogen has been
`demonstrated (P. R. Ortiz de Montellano, C. R. Wheeler, and E.
`Komives, unpublished results).
`
`Fe
`.11
`b/~
`lb
`
`Ar-c-cH•
`
`/N
`N
`
`N
`
`/
`N
`0~~·
`
`N
`
`N/
`
`Fe
`
`N
`
`N/
`
`a
`
`Ar....._ +
`C=C=o•
`•H/
`
`l~
`
`H •H
`OH
`)/...__
`Ar c/
`
`·~
`
`Ar
`
`SCHEME 1. The two consequences of cytochrome P-450-catalyzed
`arylacetylene oxidation. The square of nitrogens stands for protopor(cid:173)
`phyrin IX. The stars denote labeled atoms.
`
`hydrogen migration is coupled to metabolite formation but
`not heme alkylation makes the observation of differential
`isotope effects highly informative. Olefins are not suitable for
`such studies because the regiochemistry of oxygen addition
`which is masked in the epoxide products, can only be extracted
`by kinetic studies of secondary isotope effects. The isotope
`effects measured here for conversion of biphenylacetylene to
`biphenylacetic acid (kH/ko = 1.38) and phenylacetylene to 2-
`phenylacetic acid (kH/ko = 1.80), on the other hand, establish
`th~t enz~matic triple bond oxidation is subject to quite large
`J?nmary Isotop~ effects. T~e observed isotope effects approx(cid:173)
`Imate the maximum possible values for transition states in
`which the hydrogen moves in a sidewise rather than linear
`fashion (see Scheme 3) between the donor and acceptor atoms.
`Theoretical calculations suggest that the maximum isotope
`effect, barring tunneling effects, for a linear transition state
`(C-H-C angle= 180°) is approximately kH/kn = 7.9 whereas
`the corresponding values for angles of 1200, 90°, and 600 are
`3.0, 1.7, and 0.9, respectively (33). Experimental isotope ef(cid:173)
`fects for reactions known to involve bent transition states
`conform to these theoretical predictions (e.g. pinacol rear(cid:173)
`rangement (kH/ko = 2.7-3.3) (34), amine oxide pyrolysis (kH/
`ko = 2.7-3.2) (35), insertion of carbenes into C-H bonds (kH/
`ko = 0.9-2.5) (36, 37), 1,2-shift of a hydrogen to a vicinal
`carbene center (kH/ko = 1.7) (38), and epoxide ring cleavage
`concerted with 1,2-shift of a hydrogen (kH/ko = 1.59) (39)).
`The secondary isotope effects on a reaction in which the
`carbons undergo sp to sp2 rehybridization are, on the contrary,
`expected to be inverse and of much smaller magnitude (40).
`The primary isotope effects observed in this study are there(cid:173)
`fore, if anything, slightly larger than the cited values if allow(cid:173)
`ance is made for secondary isotope effects. Proportionately
`large intrinsic isotope effects have been demonstrated for
`~ytochrom~ P-450-catalyzed carbon hydroxylations by exper(cid:173)
`Iments which measure the competition between deuterated
`and undeuterated sites in the same molecule (43, 44), but the
`present V max isotope effects are among the largest (relative to
`the theoretical maximum) so far observed for cytochrome P-
`450.
`Oxidation of the biphenylacetylene triple bond with m(cid:173)
`chloroperbenzoic acid parallels the biological reaction in all
`respects including, as shown here, the magnitude of the kinetic
`isotope effect associated with replacement of the acetylenic
`hydrogen by deuterium. The isotope effect for the chemical
`oxidation of biphenylacetylene (kH/ko = 1.82) is larger than
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`
`Mechanism of 1r-Bond Oxidation
`
`3333
`
`1.38) but is com(cid:173)
`that for the enzymatic oxidation (kH/ko
`parable to that for the enzymatic (kH/ko 1.80) and chemical
`(kHfko = 1.73) oxidations of phenylacetylene. The isotope
`effect in the chemical reaction requires that oxygen transfer
`from the peracid to the triple bond occur in concert with
`hydrogen migration. The alternative explanation, formation
`of a quasi-stable oxirene intermediate that decomposes to the
`ketene product in a second, rate-limiting, step, is incompatible
`with the failure to detect (much less isolate) oxirene inter(cid:173)
`mediates even in frozen matrixes at cryogenic temperatures
`(32, 43, 44). The isotope effect data essentially rule out the
`formation of oxirenes in the perbenzoic acid reaction and
`consequently resolve the long standing question of whether
`oxirenes are formed in the chemical oxidation (32). The data
`substantiate the theoretically predicted asymmetry of the
`transition state for the oxidation of acetylene by peroxyformic
`acid (45). The parallel reaction course and isotope effects for
`the chemical and enzymatic reactions strongly argue, in turn,
`that oxirenes are also not intermediates in the enzymatic
`oxidation of acetylenes. Oxirene intermediates would, if any(cid:173)
`thing, be destabilized by the interactions available within the
`cytochrome P-450 active site (e.g. metal coordination, hydro(cid:173)
`gen bonding) and would therefore be even less likely to
`accumulate prior to a rate-limiting step coupled to hydrogen
`migration.
`The strong analogy between the enzymatic and m-chloro(cid:173)
`perbenzoic acid reactions contrasts with the reaction of acet(cid:173)
`ylenes with hydroxyl radicals. Hydroxyl radicals readily add
`to terminal acetylenes, including acetylene, propargyl alcohol,
`and 3-hydroxy-3-methyl-l-butyne, but the corresponding
`acids are not obtained and products compatible with hydrogen
`migration are not observed (46). Products analogous to those
`obtained in the enzymatic reaction are not obtained even if
`Cu2+ or Fe+3 is added to the Fenton system. The Fenton
`oxidation of phenylacetylene differs in that it gives a trace of
`phenylacetic acid (Fig. 6), but is generally consistent with the
`earlier work in that phenylacetic acid is but a very minor
`component of a complex mixture of products. Clearly, the
`two-electron oxidation of acetylenes mediated by m-chloro(cid:173)
`perbenzoic acid, which exclusively yields the product obtained
`in the enzymatic reaction, better models cytochrome P-450
`catalysis than the corresponding reaction with hydroxyl rad(cid:173)
`icals.
`The sharp contrast between the large isotope effects on
`metabolite formation and the absence of a detectable isotope
`effect on cytochrome P-450 destruction requires the oxidative
`trajectories that result in metabolite formation and enzyme
`inactivation to diverge prior to the point where the hydrogen
`shifts in the former pathway. This conclusion specifically
`rules out destruction of the enzyme by the ketene or any other
`species subsequent to migration of the hydrogen. The differ(cid:173)
`ential isotope effects are consistent with the fact that the
`oxygen finishes on the terminal carbon in the metabolite and
`the internal carbon in the heme adduct (7, 24, 25).
`The oxidation of asymmetrically substituted acetylenes can,
`in principle, yield two a-ketocarbenes that are formally re(cid:173)
`lated to a common oxirene (Scheme 2) (32). If the a-ketocar(cid:173)
`benes are not interconvertible via the oxirene, one a-ketocar-
`
`bene isomer could give rise to the ketene metabolite and the
`other could alkylate the prosthetic heme. The a-ketocarbenes,
`however, can only be involved in the enzymatic reaction if a(cid:173)
`ketocarbene formation is followed by a rate-determining hy(cid:173)
`drogen shift in the pathway to metabolites. The required
`formation of a relatively stable carbene intermediate is feasi(cid:173)
`ble in view of the chemical synthesis of stable carbene-iron
`complexes (47-50) and of the fact that such structures have
`been postulated for the stable complexes formed in the reac(cid:173)
`tions of cytochrome P-450 with methylenedioxyphenyl com(cid:173)
`pounds and halocarbons (51-53). Carbene complexes are read(cid:173)
`ily detected in microsomal incubations because they have a
`characteristic absorption in the 440-490 nm range. We have
`recently demonstrated that fairly stable complexes with an
`absorption maximum at 445 nm are formed in the reactions
`of cytochrome P-450 with ethyl diazoacetate and diazoaceto(cid:173)
`phenone.4 The carbene from diazoacetophenone is identical
`to that expected if a carbene intermediate is generated by
`oxygen addition to the internal carbon of phenylacetylene.
`The microsomal oxidation of phenylacetylene, however, is not
`accompanied by the detectable formation of a species that
`absorbs in the 445 nm region. 5 The absence of such a chro(cid:173)
`mophore, in view of the fact that the chromophore is readily
`detected when the expected carbene is generated by an alter(cid:173)
`native procedure (from the diazoketone), makes the interven(cid:173)
`tion of a-ketocarbene intermediates unlikely.
`The mechanism for enzymatic carbon-carbon triple bond
`oxidation must adhere to the following constraints: (a) con(cid:173)
`current ketene formation and heme alkylation, (b) quantita(cid:173)
`tive shift of the acetylenic hydrogen in the ketene pathway,
`(c) large primary isotope effects on metabolite formation but
`not heme alkylation, (d) incorporation of one atom of molec(cid:173)
`ular oxygen into the metabolite, (e) location of the oxygen on
`the terminal carbon in metabolites but on the internal carbon
`in heme adducts, and (/) strong parallels in the reactions of
`acetylenes with cytochrome P-450 and peracids but not hy(cid:173)
`droxyl radicals. These results require heme alkylation and
`metabolite formation to diverge prior to (or during) transfer
`of the oxygen to the terminal carbon for metabolite formation
`and the internal carbon for heme alkylation. The preference
`for oxygen transfer to the terminal carbon is shown by the
`absence of products from oxygen addition to the internal
`carbon in the analogous oxidation by m-chloroperbenzoic
`acid. The substrate could, however, be bound a fraction of the
`time in an orientation that forces oxygen transfer to the
`internal carbon. The relatively loose binding of substrates by
`cytochrome P-450 required for the observation of high intra(cid:173)
`molecular isotope effects (41, 42) suggests that a more com(cid:173)
`plicated mechanism may be involved. One alternative is for
`enzymatic electron transfer from the 1r·bond to precede car(cid:173)
`bon-oxygen bond formation in the heme alkylation pathway
`(Scheme 3, upper mechanism). The partitioning of substrates
`between metabolism and heme alkylation would be deter(cid:173)
`mined by the ratio of electron transfer to direct oxygen
`addition. A second alternative is for the iron-oxo complex to
`add to the phenylacetylene 1r-bond to form the two possible
`metallooxocyclobutene isomers (Scheme 3, lower mechanism).
`Rearrangement of the isomer with the phenyl vicinal to the
`iron would result in ketene formation whereas internal ligand
`transfer in the isomer with the phenyl vicinal to the oxygen
`would result in heme alkylation. The isotope effect on metab(cid:173)
`olite formation requires, however, reversible formation of the
`Ar~
`H
`SCHEME 2. The theoretical relationship between the two carbenes
`and the oxirene that could result from oxidation of an arylacetylene.
`
`4 P.R. Ortiz de Montellano and E. A. Komives, unpublished results.
`6 A search for long wavelength absorption in incubations of phen(cid:173)
`ylacetylene with liver microsomes from phenobarbital-induced rats
`has been fruitless.
`
`Boehringer Ex. 2017
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 4
`
`

`
`3334
`
`Mechanism of rr-Bond Oxidation
`
`N/N F.fL N/N . -N/N r N/ -
`
`•
`Ar--
`
`o-
`Ar -t..:
`~
`N-----N
`Felli/
`/
`N~N
`o, J
`p
`Aif
`
`[N/N 7\ N/Nl
`~ ~
`'==(
`'-H +
`
`N
`
`/
`/
`Fem
`N-----N
`+Ar..,
`C
`c== -=
`0
`w'
`
`14. Wade, A., Symons, A. M., Martin, L., and Parke, D. V. (1980)
`Biochem. J. 188, 867-872
`15. Wade, A., Symons, A.M., Martin, L., and Parke, D. V. (1979)
`Biochem. J. 184, 509-517
`16. Sullivan, H. R., Roffey, P., and McMahon, R. E. (1979) Drug
`Metab. Dispos. 7, 76-80
`17. Ortiz de Montellano, P. R., and Kunze, K. L. (1980) J. Am. Chem.
`Soc. 102, 7373-7375
`18. Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Arch.
`Biochem. Biophys. 209, 710-712
`19. McMahon, R. E., Turner, J. C., Whitaker, G. W., and Sullivan,
`H. R. (1981) Biochem. Biophys. Res. Commun. 99,662-667
`20. El Masri, A.M., Smith, J. N., and Williams, R. T. (1958) Biochem.
`J. 68, 199-204
`21. White, I. N. H., and Muller-Eberhard, U. (1977) Biochem. J.
`166,57-64
`22. Ortiz de Montellano, P.R., Kunze, K. L., Yost, G. S., and Mico,
`B. A. (1979) Proc. Natl. Acad. Sci. U.S. A. 76, 746-749
`23. Ortiz de Montellano, P. R., and Kunze, K. L. (1980) J. Bioi.
`Chem. 255, 5578-5585
`24. Ortiz de Montellano, P.R., and Kunze, K. L. (1981) Biochemistry
`20, 7266-7271
`25. Ortiz de Montellano, P. R., Beilan, H. S., and Mathews, J. M.
`(1982)J. Med. Chem. 25,1174-1179
`26. Ortiz de Montellano, P. R., Mico, B. A., Mathews, J. M., Kunze,
`K. L., Miwa, G. T., and Lu, A. Y. H. (1981) Arch. Biochem.
`Biophys. 210,717-728
`27. Orton, T. C., and Parker, G. L. (1982) Drug Metab. Dispos. 10,
`110-115
`28. Gibson, G. G., Orton, T. C., and Tamburini, P. P. (1983) Biochem.
`J. 203, 161-168
`29. Alvares, A. P., and Kappas, A. (1977) J. Biol. Chem. 252, 6373-
`6378
`30. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J.
`Bioi. Chem. 256, 1044-1052
`31. Ogata, Y., Sawaki, Y., and Inoue, H. (1973) J. Org. Chem. 38,
`1044-1045
`32. Lewars, E. G. (1983) Chem. Rev. 83, 519-534
`33. More O'Ferrall, R. A. (1970) J. Chem. Soc. Sect. B 785-790
`34. Collins, C. J., Rainey, W. T., Smith, W. B., and Kaye, I. A. (1959)
`J. Am. Chem. Soc. 81,460-466
`35. Chiao, W.-B., and Saunders, W. H. (1978) J. Am. Chem. Soc.
`100, 2802-2805
`36. Simons, J. W., and Rabinovitch, B. S. {1963) J. Am. Chem. Soc.
`85, 1023-1024
`37. Goldstein, M. J., and Dolbier, W. R. (1965) J. Am. Chem. Soc.
`87, 2293-2295
`38. Kyba, E. P., and Hudson, C. W. (1977) J. Org. Chem. 42, 1935-
`1939
`39. Gillilan, R. E., Pohl, T. M., and Whalen, D. L. (1982) J. Am.
`Chem. Soc. 104, 4482-4484
`40. Halevi, E. A. (1963) Prog. Phys. Org. Chem. 1, 109-221
`41. Hjelmeland, L. M., Aronow, L., and Trudell, J. R. (1977) Biochem.
`Biophys. Res. Commun. 76, 541-549
`42. Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J.
`(1978) Biochem. Biophys. Res. Commun. 81, 154-160
`43. Torres, M .• Bourdelande, J. L., Clement, A., and Strausz, 0. P.
`(1983) J. Am. Chem. Soc. 105, 1698-1700
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`(1983) J. Am. Chem. Soc. 105, 7457-7459
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`53. Elcombe, C. R., Bridges, J. W., Gray, T. J. B., Nimmo-Smith, R.
`
`t
`/¥·/r./=-/7
`
`N----N
`
`N-----N
`
`Nr----«
`
`O_#H
`
`o..
`
`O#Ar
`
`H
`A r - -
`Ar
`SCHEME 3. Two mechanisms for triple bond oxidation consistent
`with the available information. In the upper half of the scheme is a
`mechanism in which heme alkylation results from initial electron
`transfer and metabolite formation from direct oxygen transfer. In the
`bottom half is a mechanism involving the reversible formation of
`isomeric metallooxocyclobutenes.
`
`metallooxocyclobutene isomers followed by rate-determining
`hydrogen migration in the pathway to metabolites. Metal(cid:173)
`looxocyclobutane intermediates, first proposed by Sharpless
`et al. (54) tQ explain the oxidation of olefins by chromyl
`chloride, have been observed by NMR in the reaction of
`osmium tetroxide with 1,1-diphenylethylene (55) and have
`been invoked by Collman et al. (56) to explain olefin oxidation
`by an oxo-manganese complex. No precedent is yet available,
`however, for the formation of metallooxocyclobutene inter(cid:173)
`mediates with iron porphyrins