Fpl 2011 Poraj-Kobielska001.Pdf

Biochemical Pharmacology 82 (2011) 789–796

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Biochemical Pharmacology

jo urnal homepage: www.elsevier.com/locate/biochempharm

Preparation of human drug metabolites using fungal peroxygenases

a a, a b

Marzena Poraj-Kobielska , Matthias Kinne *, Rene´ Ullrich , Katrin Scheibner ,

a c a

Gernot Kayser , Kenneth E. Hammel , Martin Hofrichter

International Graduate School of Zittau (IHI Zittau), Department of Bio- and Environmental Sciences, Markt 23, 02763 Zittau, Germany

Section of Biotechnology, Chemistry and Process Engineering, Lausitz University of Applied Sciences, Großenhainer Straße 57, 01968 Senftenberg, Germany

USDA Forest Products Laboratory, Madison, WI 53726, USA, and Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA

A R T I C L E I N F O A B S T R A C T

Article history: The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective

Received 28 March 2011

monooxygenation remains a challenging task for synthetic organic chemists. Here we report that

Accepted 14 June 2011

aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and

Available online 23 June 2011

Coprinellus radians catalyzed the H2O2-dependent selective monooxygenation of diverse drugs, including

acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenaﬁl and tolbutamide. Reactions

Keywords:

included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations

Peroxidase

and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs

Peroxygenation

were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of Hydroxylation

O-Dealkylation tolbutamide, acetanilide and carbamazepine in the presence of H2 O2 resulted in almost complete

N-Dealkylation incorporation of O into the corresponding products, thus establishing that these reactions are

Cytochrome P450 peroxygenations. The deethylation of phenacetin-d1 showed an observed intramolecular deuterium

isotope effect [(kH/kD)obs] of 3.1 0.2, which is consistent with the existence of a cytochrome P450-like

intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful

biocatalytic tools to prepare pharmacologically relevant drug metabolites.

1. Introduction reaction rates [7,8]. More promising results have been reported

for laboratory-evolved bacterial P450s, which are capable of

Human drug metabolites (HDMs) are valuable chemicals catalyzing the H2O2-dependent hydroxylation of pharmaceuti-

needed for the development of safe and effective pharmaceuticals. cals via the ‘‘peroxide shunt’’ pathway [9]. Recent studies,

They are frequently used as substrates and authentic standards in however, have demonstrated that this approach needs further

studies of drug bioavailability, pharmacodynamics and pharma- optimization [10]. Alternatively, modiﬁed hemoproteins such

cokinetics [1–3]. In vivo, the C–H bonds of pharmaceuticals are as microperoxidases might be used to catalyze hydroxylations

predominantly oxygenated by liver cytochrome P450-monoox- by a P450-like oxygen transfer mechanism, but so far these

ygenases (P450s) to yield more polar HDMs that are excreted catalysts do not exhibit the necessary performance and

directly or as conjugates [4]. This directed incorporation of an selectivity [11–16].

oxygen atom into a complex organic structure is one of the most Here we have adopted a recently developed approach, using

challenging reactions in synthetic organic chemistry [5] and thus aromatic peroxygenases (APOs) from the agaric basidiomycetes

low yields and the need for laborious removal of byproducts have Agrocybe aegerita (AaeAPO) and Coprinellus radians (CraAPO) to

prevented the cost-effective preparation of HDMs by purely produce diverse HDMs. These stable, secreted enzymes oxidize a

chemical methods [6]. wide range of substrates and are promising oxidoreductases for

Another approach is the in vitro preparation of HDMs with biotechnological applications [6,17–23].

enzymes. The obvious route is to use isolated human P450s, but

these complex multiprotein systems are membrane-bound,

poorly stable, cofactor-dependent, and generally exhibit low

In previous publications, the enzymes were also referred to as haloperoxidase-

peroxygenases, mushroom/fungal peroxygenases, AaP (Agrocybe aegerita peroxi-

dase/peroxygenase) or CrP (Coprinellus radians peroxygenase) [5,18,20,22]. In

* Corresponding author. Tel.: +49 3583612723; fax: +49 3583612734. February 2011, they were classiﬁed in the Enzyme Nomenclature under EC 1.11.2.1

E-mail address: [email protected] (M. Kinne). (unspeciﬁc peroxygenase).

790 M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796

2. Materials and methods ammonium formate (pH 3.5)/acetonitrile, 95:5 for 5 min, followed

by a 25-min linear gradient to 100% acetonitrile. For the

2.1. Reactants experiments with acetaminophen, acetanilide and diclofenac, a

Synergi 4 m Fusion RP-80A column (4.6 mm diameter by 150 mm

Metoprolol, oseltamivir phosphate, 4-hydroxytolbutamide, 4 - length, 4 mm particle size, Phenomenex) was used. The column

hydroxydiclofenac, 5-hydroxydiclofenac, 3-hydroxyacetamino- was eluted at 40 8C and a ﬂow rate of 1 ml min with a mixture of

phen, omeprazole and sildenaﬁl were obtained from Chemos GmbH aqueous phosphoric acid solution (15 mM, pH 3) and acetonitrile,

(Regenstauf, Germany). 3-Hydroxycarbamazepine, 1-hydroxyibu- 95:5, for 5 min, followed by a 20-min linear gradient to 100%

profen, 2-hydroxyibuprofen, 1-oxoibuprofen and N-desmethylsil- acetonitrile. For metoprolol, a Gemini-Nx 3 m 110A C18 reverse

denaﬁl were purchased from Toronto Research Chemicals, Inc. phase column (2 mm diameter by 150 mm length, 3 mm particle

(Toronto, Canada), a-hydroxymetoprolol, glycinexylidide and size, Phenomenex) was used. The column was eluted at 45 8C and a

monoethylglycinexylidide from TLC PharmaChem., Inc. (Vaughan, ﬂow rate of 0.3 ml min with a mixture of aqueous 0.2% vol/vol

Canada). 17a-Ethinylestradiol, (R)-naproxen, 4-hydroxyproprano- ammonium (pH 10.5) and acetonitrile, 95:5, for 1 min, followed by

lol, 5-hydroxypropranolol, (S)-N-desisopropylpropranolol and O- a 20-min linear gradient to 80% acetonitrile, followed by 21-min

desmethylmetoprolol were obtained from Fluka (St. Gallen, linear gradient to 100% acetonitrile. Chiral separation was done

Switzerland), Shanghai FWD Chemicals, Ltd. (Shanghai, China), using the HPLC apparatus above, but using a Whelk-O 5/100

Biomol GmbH (Hamburg, Germany), SPIbio, Bertin Group (Montigny Kromasil column (4.6 mm diameter by 250 mm length, Regis

Le Bretonneux, France), ABX Advanced Biochemical Compounds Technologies (Morton Grove, USA). The isocratic mobile phase

(Radeberg, Germany) and Sandoo Pharmaceuticals & Chemicals Co., consisted of 80% vol/vol methanol and 20% of 15 mM phosphate

18 1

Ltd. (Zhejiang, China), respectively. H2 O2 (90 atom%, 2% wt/vol) buffer. The columns were operated at 40 8C and 1 ml min for

was a product of Icon Isotopes (New York, USA). All other chemicals 30 min. Retention times for products are given in Supplemental

were purchased from Sigma-Aldrich (Schnelldorf, Germany). Table 1.

Phenacetin-d1 (N-(4-[1- H]ethoxyphenyl)acetamide) was pre- Aliphatic aldehydes were analyzed as their 2,4-dinitrophenyl-

pared from acetaminophen (N-(4-hydroxyphenyl)acetamide) and hydrazones after addition of 0.2 volume of 0.1% 2,4-dinitrophe-

ethyl iodide-d1 as described previously for phenacetin-d3 [24]. The nylhydrazine solution in 0.6 N HCl to each reaction mixture. The

reaction product of the synthesis (phenacetin-d1) was identiﬁed by derivatized products were analyzed using the same HPLC

comparison of retention time, UV absorption spectra, and mass apparatus as above, but the Luna C18 column was eluted with

spectra relative to authentic phenacetin [25]. Mass spectrum (m/z, aqueous 0.1% vol/vol ammonium formate (pH 3.5)/acetonitrile,

%): 180 (100, H), 138 (62, C2H2O), 110 (12, C4DH6O), 109 (96, 70:30 for 5 min, followed by a 20-min linear gradient to 100%

C3DH2O2), 108 (100, C3DH6ON), 80 (16) and 43 (18). acetonitrile. Stoichiometrical experiments on sildenaﬁl N-demeth-

The extracellular peroxygenase of A. aegerita (AaeAPO; isoform ylation were conducted in stirred reactions (0.20 ml) that

II, 45 kDa) was produced and puriﬁed as described previously [26]. contained 2 U ml (0.144 mM) peroxygenase, potassium phos-

The enzyme preparation was homogeneous by sodium dodecyl phate buffer (50 mM, pH 7.0) and the substrate (0.25 mM). The

polyacrylamide gel electrophoresis (SDS-PAGE) and exhibited an reactions were initiated with 0.0023–0.037 mM H2O2.Mass

A418/A280 ratio of 1.75. The speciﬁc activity of the AaeAPO spectroscopic determinations were made in positive or negative

preparation was 117 U mg , where 1 U represents the oxidation ESI mode (electrospray ionization) in a mass range from 70 to 500,

of 1 mmol of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to 3,4- step size 0.1, drying gas temperature 350 8C, capillary voltage

dimethoxybenzaldehyde (veratraldehyde) in 1 min at 23 8C [26]. 4000 V for positive mode and 5500 V for negative mode. The

The Coprinellus radians peroxygenase (CraAPO) was produced and reaction products were identiﬁed relative to authentic standards,

puriﬁed as described previously [27]. It was homogeneous by SDS- based on their retention times, UV absorption spectra, and mass

PAGE, exhibited an A419/A280 ratio of 1.04 and had a speciﬁc activity spectral [M+H] or [MH] ions.Quantiﬁcations of reactants and

of 25.8 U mg in the above assay. products were obtained by HPLC as described above, using a linear

external standard curve (R > 0.98) of the respective compound.

2.2. Product identiﬁcation The parameters were calculated as follows: total conversion

[(Sc Ss)/Sc) 100%; yield: (P/Sc) 100%; regioselectivity: (P/

Typical reaction mixtures (0.2–1.0 ml) contained puriﬁed (Sc Ss)] 100% (Sc is the substrate concentration of the control,

peroxygenase (1.0–2.0 U ml = 0.4–0.8 mM), substrate to be Ss is the substrate concentration after reaction with the enzyme

oxidized (0.5–2.0 mM), potassium phosphate buffer (50 mM, pH and P is the product concentration).

7.0) and ascorbic acid (4.0–6.0 mM, to inhibit further oxidation of

any phenolic products that were released [6,17]). The reactions 2.3. Enzyme kinetics

were started by the addition of limiting H2O2 (2.0–4.0 mM) and

stirred at room temperature. They were stopped by addition of The kinetics of propranolol hydroxylation as well as metoprolol

sodium azide (1 mM) or trichloroacetic acid (TCA) (5%) after 3 min. and phenacetin O-dealkylation were analyzed in stirred microscale

In some cases, H2O2 was added continuously with a syringe pump reactions (0.10 ml, 23 8C) that contained 0.40 mM peroxygenase

and the reactions were stopped after 4–6 h, when chromatograph- (AaeAPO), potassium phosphate buffer (50 mM, pH 7.0), ascorbic

ic analyses showed that product formation was complete. acid (4 mM) and 0.010–5.000 mM substrate. The reactions were

Reaction products of interest were obtained, generally with initiated with 2.0 mM H2O2. Reactions with propranolol or

baseline resolution, by high performance liquid chromatography phenacetin were stopped with 0.010 ml of 50% TCA solution after

(HPLC) using an Agilent Series 1200 instrument equipped with a 5 s, and reactions with metoprolol were stopped with 0.2 volume

diode array detector (DAD) and an electrospray ionization mass of 0.1% 2,4-dinitrophenylhydrazine solution in 0.6 N HCl after 10 s.

spectrometer (MS) (Agilent Technologies Deutschland GmbH, The resulting products were quantiﬁed by HPLC as described above

Bo¨blingen, Germany). Unless otherwise stated, reverse phase using external standard curves prepared with authentic standards.

(RP) chromatography of reaction mixtures was performed on a The kinetics of acetanilide hydroxylation were analyzed in

Luna C18 column (2 mm diameter by 150 mm length, 5 mm stirred reactions (0.5 ml, 23 8C) that contained 0.08 mM AaeAPO,

particle size, Phenomenex (Aschaffenburg, Germany), which was potassium phosphate buffer (50 mM, pH 7.0), ascorbic acid (4 mM)

eluted at 0.35 ml min and 40 8C with aqueous 0.01% vol/vol and 0.010–5.000 mM substrate. The reactions were initiated with

M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796 791

2.0 mM H2O2 and stopped with 0.050 ml of sodium azide solution Using AaeAPO and two of the above substrates, we investigated

(1 mM) after 5 s. The initial velocity of acetaminophen formation the source of the oxygen introduced during hydroxylation. When

was then determined from the increase in absorbance at 290 nm the oxidation of acetanilide (II) was conducted with H2 O2 in

1 1

(e = 1100 M cm ) using a Cary 50 UV/visible spectrophotome- place of H2O2, mass spectral analysis of the resulting acetamino-

ter. The kinetic parameters were determined by nonlinear phen showed that the principal [MH] ion had shifted from its

regression using the Michaelis–Menten model in the ANEMONA natural abundance m/z of 150 to m/z 152. Likewise, the

program [28]. hydroxylation of 3-hydroxycarbamazepine under these conditions

resulted in a shift of the [M+H] ion from its natural abundance m/z

2.4. O-labeling experiments of 253 to m/z 255. Accordingly, H2O2 was the source of the

introduced oxygen in both reactions.

The reaction mixtures (0.20 ml, stirred at room temperature) Also using AaeAPO, we determined apparent kinetic constants

contained 2 U ml (0.08 mM) of AaeAPO, potassium phosphate for two of these aromatic hydroxylations. For propranolol, the Km

buffer (50 mM, pH 7.0) and 0.5 mM substrate (tolbutamide, was 442 mM and the kcat was 59 s ; for acetanilide, the constants

acetanilide, or carbamazepine). Reactions with tolbutamide or were 1,310 mM and 1,925 s .

acetanilide were initiated with a single addition of 2.0 mM (ﬁnal

concentration) H2 O2. For reactions with carbamazepine, the 3.2. Aliphatic hydroxylation

same quantity of H2 O2 was added continuously with a syringe

pump over 6 h. A portion of each completed reaction was then Both peroxygenases catalyzed the hydroxylation of aliphatic

analyzed by HPLC/MS as described in Section 2.2. For each m/z side chains. Thus, ibuprofen (IV) was predominantly oxidized to 2-

value, the average total ion count within the metabolite (4- hydroxyibuprofen, with the CraAPO-catalyzed reaction exhibiting

hydroxytolbutamide, acetaminophen and 3-hydroxycarbamaze- a higher yield and regioselectivity. Similarly, tolbutamide (VII) was

pine) peak was used after background correction to generate the oxidized to 4-hydroxytolbutamide (Table 1). A mass spectral

ion count used for mass abundance calculations [18]. experiment with tolbutamide and H2 O2 as the substrates showed

that the resulting 4-hydroxytolbutamide exhibited a principal

2.5. Determination of intramolecular isotope effect [MH] ion with an m/z of 289 rather than 287, again showing that

H2O2 was the source of the introduced oxygen (Fig. 3).

The reaction mixture (0.2 ml) contained puriﬁed AaeAPO

(2 U ml , 0.08 mM), potassium phosphate buffer (50 mM, pH 3.3. O-Dealkylation

7.0), phenacetin-d1 and ascorbic acid (4.0 mM). The reaction was

started by the addition of H2O2 (2.0 mM) and stirred at room Both APOs cleaved alkyl aryl ether linkages in various drugs to

temperature. The reaction was stopped by the addition of 10% yield phenols [18]. Thus, metoprolol (VIII) was selectively

sodium azide (10 mM) after 10 s. The reaction products were demethylated to O-desmethylmetoprolol, naproxen (IX) to O-

analyzed as described above. For each m/z value, the average total desmethylnaproxen and dextromethorphan (X) to dextrorphan.

ion count within the acetaminophen peak was used after Yields and regioselectivities were generally greater with AaeAPO.

background correction to generate the ion count used for mass The above phenolic reaction products all tended to undergo further

abundance calculations. oxidation to quinones and/or coupling products because APOs

exhibit general peroxidase activity that generates phenoxyl

3. Results radicals from phenols [5,6,17,18], but this undesired reaction

was partially inhibitable via addition of the radical scavenger

The peroxygenases from Agrocybe aegerita (AaeAPO) and ascorbate to the reactions. DNPH derivatization of the reaction

Coprinellus radians (CraAPO) oxidized diverse pharmaceuticals mixtures showed that the methyl group was released as

(I–XX, Fig. 1), showing different selectivity in some cases. The formaldehyde in each case. A kinetics analysis done with AaeAPO

products, most of which we obtained with baseline resolution by and one of the substrates, metoprolol, showed that the reaction

HPLC, were monooxygenated or dealkylated compounds that exhibited an apparent Km for it of 2,330 mM and an apparent kcat of

correspond in most cases to previously identiﬁed HDMs (Table 1). 96 s .

Below, we have classiﬁed the major reactions according to the Analogously to the demethylations discussed above, phenace-

chemical moiety that was oxidized on the targeted drug. In many tin (XI) was de-ethylated to give acetaminophen and acetaldehyde

cases, a single oxidation predominated but, as noted in Table 1, (Table 1). Since phenacetin has a symmetrical site at its a-carbon, it

more than one of these reactions sometimes occurred on a single is a suitable substrate to determine whether a catalyzed

substrate. A complete overview of all reactants investigated, etherolytic reaction exhibits an intramolecular deuterium isotope

conﬁrmed products and hypothetical products for which we lacked effect. LC/MS analysis of DNPH-derivatized reactions showed that

authentic standards (drawn in brackets), is presented in Supple- the AaeAPO-catalyzed cleavage of N-(4-[1- H]ethoxyphenyl)ace-

mentary Table 2. tamide (phenacetin-d1) resulted in a preponderance of [ H]acetal-

dehyde 2,4-dinitrophenylhydrazone (m/z 224, [MH] ) over

3.1. Aromatic hydroxylation natural abundance acetaldehyde 2,4-dinitrophenylhydrazone

(m/z 223, [MH] ) (Fig. 4). The observed mean intramolecular

Both APOs catalyzed the hydroxylation of aromatic rings, in deuterium isotope effect [(kH/kD)obs] from three experiments was

some cases with high regioselectivity. For example, AaeAPO 3.1 0.2. The apparent Km of AaeAPO for phenacetin was 998 mM and

preferentially oxidized propranolol (I) to 5-OH-propranolol [17], the apparent kcat was 33 s .

acetanilide (VI) to acetaminophen (paracetamol; Fig. 2), carba-

mazepine (III) to 3-OH-carbamazepine and diclofenac (IV) to 4 - 3.4. N-Dealkylation

OH-diclofenac [17]. CraAPO catalyzed the same reactions but with

lower regioselectivity. Both enzymes also oxidized tamoxifen (V) The APOs catalyzed the oxidative N-dealkylation of several

to produce low yields of 4-OH-tamoxifen and other products, drugs that contain secondary or tertiary amine groups. For

which we were unable to resolve sufﬁciently by HPLC to permit example, AaeAPO regioselectively N-dealkylated sildenaﬁl (XII)

quantiﬁcations. at the tertiary N in its N-methyl piperazine ring to give N-

792 M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796 I Propranolol II Acetanilide III Carbamazepin e IV Diclofenac V Tamoxifen (ß-blocker) (anti-inflammatory) (anticonvulsant) (anti-inflammatory) (anti-cancer) N H N O O N 2 O Cl OH H O NH N NH Cl OH

O VIIbuprofen VII Tolbutamide (anti-inflammatory) (Na+ -channel blocker) IX Naproxen O VIII Metoprolol OH OO (anti-inflammatory) S (ß-blocker)

O NH NH O OH

XDextromethorphan ONHO O (antitussive ) XI Phenacetin OH (anti-inflammatory) N XII Sildenafil XIIILidocaine H NH (neuraminidase inhibitor) (anesthetic) O O O N N N O O N O N N NH

N O XIV 4-Dimethylaminoantipyrine H O (antipyrretic) XV Oseltamivir (antiviral) XVII Sulfadiazine N (antibacterial) XVI Ketoprofen O N (anti-inflammatory) NH O S N N O O O O O O N H N NH 2 O OH H N 2 XXI Felbamate XX Chlorpromazine XVIII Gemfibrozil (anticonvulsant)

(lipid-lowering agent) (antipsychotic) XIX Omeprazol e

Cl O (proton pump inhibitor) NH2 O OOH N O O S O O N SN O NH XXII H 2 17α -Ethinylestradiol N N O (contraceptive) XXIII Reichstein substance S OH (prohormone) HO O H OH H H HO

Fig. 1. Chemical structures of pharmaceuticals tested as peroxygenase substrates.

desmethylsildenaﬁl and formaldehyde in high yield. By contrast, glycinexylidide and of 4-dimethylaminoantipyrine (XIV) to 4-

CraAPO was ineffective in this oxidation (Table 1). A quantitative aminoantipyrine.

analysis of AaeAPO-catalyzed sildenaﬁl oxidation in the presence

of the limiting H2O2 showed that one equivalent of N-desmethyl- 3.5. Ester cleavage

sildenaﬁl was formed per equivalent of oxidant supplied (Table 2).

Among the other N-dealkylations listed in Table 1 are the Ester cleavage by a peroxygenase can be regarded as a special

conversion of lidocaine (XIII) to monoethylglycinexylidide and case of O-dealkylation. Thus, CraAPO selectively cleaved oselta-

M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796 793

Table 1

Products identiﬁed by mass spectroscopy after oxidation of pharmaceuticals by AaeAPO and CraAPO in the presence of H2O2. The m/z value for the major observed diagnostic

ion, consumed substrate (SC), the yield (Y) and the regioselectivity (RS) of the product is shown in each case.

Substrate Reaction product Discussed as HDM m/z AaeAPO (%) CraAPO (%)

in Ref no.

SC Y RS SC Y RS

I 23 11

5-OH-Propranolol [9,29] [M+H] 276 21 91 6 53

4-OH-Propranolol [9,29,46] [M+H] 276 – 2 13

N-Desisopropyl-propranolol [9,29] [M+H] 218 Traces 3 22

II 89 20

Acetaminophen [47–49] [MH] 150 80 90 13 65

3-OH-Acetaminophen [47] [MH] 166 5 5 1 2

III 22 15

3-OH-Carbamazepine [50] [M+H] 253 13 61 6 40

IV 78 15

0 +

4 -OH-Diclofenac [51,52] [M+H] 312 68 87 5 30

V 25

4-OH-Tamoxifen [53–55] [M+H] 388 n.d. n.d.

N-Desmethyltamoxifen [53,54] [M+H] 358 n.d. n.d.

Endoxifen [53,54] [M+H] 374 n.d. n.d.

VI 87 98

2-OH-Ibuprofen [56,57] [M+H] 223 21 24 74 75

1-OH-Ibuprofen [57] [M+H] 223 7 8 –

1-Oxo-Ibuprofen n.a. [M+H] 221 Traces –

VII 25 20

4-OH-Tolbutamide [58] [MH] 287 15 60 13 62

VIII 82 73

O-Desmethylmetoprolol [59] [M+H] 254 17 20 4 5

a-OH-Metoprolol [59] [M+H] 284 2 2 1 1

IX 60 10

O-Desmethylnaproxen [60,61] [MH] 215 57 95 9 85

X 17 15

Dextrorphan [62,63] [M+H] 258 16 95 8 53

XI 34 16

Acetaminophen [64,65] [M+H] 152 23 66 13 80

3-OH-Acetaminophen [64] [M+H] 168 2 5 –

XII 82 80

N-Desmethylsildenaﬁl [66] [M+H] 461 82 99 4 5

XIII 60 45

Monoethylglycinexylidide [67] [M+H] 207 25 41 32 70

Glycinexylidide [67] [M+H] 179 18 30 5 11

XIV 68 38

4-Aminoantipyrine [68] [M+H] 204 16 23 19 48

XV – 80

Oseltamivir carboxylate [69] [M+H] 285 – 71 88

Mass spectral data indicate the formation of corresponding carbonyls as described previously [19] (see supplementary Material).

A syringe pump was used for hydrogen peroxide supply. (n.d.) = not determined due to poor resolution; () = not detected; (n.a.) = not applicable.

mivir (XV) to oseltamivir carboxylate and acetaldehyde in high neither one predominated in the end-product mixture (data not

yield. Interestingly, oseltamivir was not converted by AaeAPO. shown).

3.6. Additional observations 4. Discussion

For some products of APO-catalyzed oxidations no authentic Our results show that two fungal peroxygenases (APOs)

standards were available. However, the mass shifts we observed catalyzed the hydroxylation or dealkylation of diverse pharma-

after these reactions (see supplementary material) indicate the ceuticals, in some cases with high regioselectivity. Furthermore,

regioselective oxidation of ketoprofen (XVI), sulfadiazine (XVII), most of the oxidations matched those catalyzed by human liver

gemﬁbrozil (XVIII), omeprazole (XIX) and chlorpromazine (XX). P450s, e.g. propranolol to 5-hydroxypropranolol (CYP2D6) [29],

We also noted some limitations on substrates for the APOs. No tolbutamide to 4-hydroxytolbutamide (CYP2C9) [30], naproxen

product formation was observed from felbamate (XXI), 17a- to O-desmethylnaproxen (CYP1A2) [31], acetanilide to acet-

ethinylestradiol (XXII), or Reichstein substance S (XXIII). More- aminophen (CYP1A2) [31,32] and sildenaﬁl to N-desmethylsil-

over, we found that the APOs generally failed to discriminate denaﬁl (CYP3A4) [33]. The catalyzed reactions: aromatic

between chiral centers in the pharmaceutical substrates. For hydroxylation, aliphatic hydroxylation, O-dealkylation of ethers

example, chiral HPLC separation of the two O-desmethylnaproxen and esters and N-dealkylation of amines, are all typical of P450s

entantiomers that resulted from naproxen oxidation showed that [34,35]. Our data agree with previous work that suggests the

794 M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796

500 Table 2

Stoichiometry of sildenaﬁl (XII) oxidation by AaeAPO.

H2O2 added N-Desmethylsildenaﬁl Ratio

400 (mM) produced (mM) N-desmethylsildenaﬁl/H2O2

O O 2.3 2.12 0.92

4.6 4.27 0.93

300 NH NH 9.2 8.75 0.96

18.3 20.49 1.12

APO 36.6 32.37 0.88

200 The initial sildenaﬁl concentration was 250 mM.

H2O2 H2O

Concentration (μM) OH

extracellular APOs share characteristics with intracellular P450s,

100

which also catalyze H2O2-dependent oxidations by a pathway

termed the peroxide shunt [36–38]. These peroxygenations are

thought to be initiated when the enzyme heme is oxidized by

H2O2 to give an iron species (oxo-ferryl iron, compound I) that

02030 10 40 200 400 600

carries one of the peroxide oxygens, which subsequently

Reaction time (s)

oxidizes a C–H bond in the substrate [39].

According to the above model, oxygen incorporation from H2O2

Fig. 2. Time course of AaeAPO-catalyzed para-hydroxylation of acetanilide (*, II) to

should be quantitative when the substrate is oxidized. Our data on

acetaminophen (&).

APO-catalyzed pharmaceutical oxidations agree with this picture

since 100% of the oxygen present in newly generated phenolic or

benzylic reaction products – e.g. in the phenol group of

acetaminophen or in the alcohol moiety of 4-hydroxytolbutamide

O 18

O – was O-labeled when the experiment was conducted with

NH NH H2 O2. Moreover, the stoichiometrical result we obtained using

HN HN

sildenaﬁl as the substrate – one equivalent of N-desmethylsilde-

S O S O

O O naﬁl formed per equivalent of H2O2 supplied – agrees with the

APO two-electron oxidation expected from a peroxygenative mecha-

VII VIIa

nism (Table 2).

HO22 Also consistent with a P450-like mechanism is the intramolec-

HO2

OH ular deuterium isotope effect we observed for phenacetin-d1

VII oxidation by AaeAPO, in that our value of (kH/kD)obs around 3 is

close to the values of (kH/kD)obs near 2 that have been observed for

VIIb the P450-catalyzed O-dealkylation of this substrate. A value of 3 is

considerably smaller than the intrinsic isotope effect near 10

VIIa expected for a hydrogen abstraction mechanism, and may indicate

that phenacetin oxidation by APOs proceeds instead via electron

m/z transfer, as proposed earlier for the P450-catalyzed reaction [40].

By contrast, the O-dealkylation of 1,4-dimethoxybenzene-d3 by

AaeAPO probably does proceed via hydrogen abstraction, because

Fig. 3. HPLC elution proﬁles after conversion of tolbutamide (VII) in the presence of it exhibits a much higher (kH/kD)obs near 12 [18,40].

ascorbic acid: (front proﬁle) control without enzyme; (background proﬁle) completed

The kinetics data we report here for AaeAPO action on a

reaction with AaeAPO. Insets of mass spectra showing the incorporation of O from

18 variety of pharmaceuticals suggest that APOs may be useful

H2 O2 into the alcohol group of 4-hydroxytolbutamide (VIIa) after hydroxylation of

alternatives to P450s for the regioselective preparation of HDMs.

tolbutamide by AaeAPO are as follows. Upper: MS of the product obtained with natural

abundance H2O2. Lower: MS of the product obtained with 90 atom% H2 O2. The peak Although some P450s are known to bind pharmaceutical

VIIb showed a mass as expected for 4-oxo-tolbutamide, a potential oxidation product substrates more strongly than APOs, exhibiting Km values between

of 4-hydroxytolbutamide (see supplemental Table 2) [19].

1 and 70 mM, they generally exhibit relatively low kcat values in the

vicinity of 0.2 s or less [40,41]. As a result, the catalytic

efﬁciencies [kcat/Km] of AaeAPO for pharmaceutical oxidations lie

in the same range as those of the P450s, as exempliﬁed by our

5 1 1

values for propranolol hydroxylation (1.32 10 M s ), meto-

4 1 1

prolol O-dealkylation (4.1 10 M s ) and phenacetin O-deal-

k 4 1 1

D kylation (3.3 10 M s ). The kcat/Km value we obtained for

6 1 1

acetanilide (1.5 10 M s ) is much higher than the corre-

Aae

APO sponding value found for the P450-catalyzed reaction [32].

APOs have some advantages over P450s, including currently

k available laboratory-evolved P450s, where reaction yields are

* concerned [17]. For example, the transformation of diclofenac to 4-

mz/ hydroxydiclofenac by mutants of P450cam (CYP101A1) resulted in

total conversions between 15 and 44% and yields around 10% [42].

Fig. 4. Cleavage of N-(4-[1- H]ethoxyphenyl)acetamide (phenacetin-d1) by AaeAPO. By contrast, wild-type AaeAPO gave a total conversion of 78% and a

The mass spectrum of the reaction mixture containing the products acetaldehyde

yield of 68% for this reaction. In addition, fungal APOs may be

2,4-dinitrophenylhydrazone ([MH] , 123) and [ H] acetaldehyde 2,4-

better than P450s in practical applications for several other

dinitrophenylhydrazone ([MH] , 126) obtained from the oxidation of

reasons: (i) they utilize an inexpensive co-substrate, H O ; (ii) they

phenacetin-d1 is shown. The mass spectrum is one of three used to calculate the 2 2

observed mean intramolecular isotope effect. do not require costly co-reactants such as pyridine nucleotides,

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