Probing the scope of the sulfoxidation activity of vanadium bromoperoxidase from Ascophyllum nodosum

Article abstract of DOI:10.1016/S0957-4166(99)00514-5

It has been shown previously that the vanadium bromoperoxidase from Ascophyllum nodosum mediates the production of (R)-methyl phenyl sulfoxide with 91% enantiomeric excess from the corresponding sulfide in the presence of hydrogen peroxide. Investigation

Full text of DOI:10.1016/S0957-4166(99)00514-5

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 4563–4572
Probing the scope of the sulfoxidation activity of vanadium
bromoperoxidase from Ascophyllum nodosum
Hilda B. ten Brink,a Herbert L. Holland,b Hans E. Schoemaker,c Hester van Lingen a and
Ron Wever a,∗
aE.C. Slater Institute, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands
bDepartment of Chemistry, Brock University, St. Catharines, Ontario, L2S 3A1, Canada
cDSM Research, Bio-Organic Chemistry, PO Box 18, 6160 MD Geleen, The Netherlands
Received 25 October 1999; accepted 15 November 1999
Abstract
It has been shown previously that the vanadium bromoperoxidase from Ascophyllum nodosum mediates the
production of (R)-methyl phenyl sulfoxide with 91% enantiomeric excess from the corresponding sulfide in the
presence of hydrogen peroxide. Investigation of the sulfoxidation activity of this enzyme shows that activating
substituents at the para-position of the aromatic ring of methyl phenyl sulfide positively influence the selectivity
of the reaction, whereas strongly electron-withdrawing groups cancel the catalyzed sulfoxidation reaction. The
first evidence is presented that the vanadium bromoperoxidase catalyzes the sulfoxidation of racemic non-aromatic
cyclic thioethers with high kinetic resolution. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Several different catalytic methods for the production of chiral sulfoxides have been studied in
great detail over the last two decades due to the growing demand for these chiral auxiliaries in
asymmetric synthesis, in particular for the preparation of biologically active compounds.1,2 In addition
to chemically based reactions3,4 great interest has developed for both whole-cell5–9 and enzymatic10–19
approaches, as more satisfying results are obtained using biological procedures. A great variety of organic
sulfides have been converted to their corresponding sulfoxides using fungi, including Helminthosporium
species NRRL 4671 and Mortierella isabellina, and moderate to high yields and enantiomeric purities
were obtained.5–9 In addition, the conversion of prochiral organic sulfides to the corresponding chiral
sulfoxides has been shown to be catalyzed by various isolated enzymes,10–13 including a large number
of different heme peroxidases,14–17 showing in general rather low enantioselectivities and turnovers. The
∗
Corresponding author. Tel: +31-20-525-5110; fax: +31-20-525-5124; e-mail: a311rw@chem.uva.nl
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00514-5
tetasy 3149

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heme chloroperoxidase from Caldariomyces fumago is an exception as this enzyme was shown to be
capable of catalyzing oxygen transfer reactions with very high enantioselectivity and versatility.18,19
However, the potential application of this class of peroxidases in asymmetric organic synthesis is
hindered, primarily due to the gradual oxidative inactivation of these enzymes by their natural co-
substrate, hydrogen peroxide, during catalysis.20,21
Recently, it was shown that another class of peroxidases, which contain a vanadate ion in the active
site as the prosthetic group, is capable of selectively mediating the transformation of aromatic organic
sulfides to the corresponding sulfoxides.22,23 Previously, these vanadium haloperoxidases were thought
to oxidize only halides (X−) in the presence of hydrogen peroxide yielding hypohalous acid (HOX),
a highly reactive intermediate, which in turn reacts with an electrophilic acceptor, if present, to form
a halogenated compound. In contrast to the high selectivity of the sulfoxidation reactions catalyzed by
these enzymes, mediation of selective halogenation reactions has not been established for these enzymes
except for one observation of regioselective bromohydration of certain cinnamyl substrates.24 Moreover,
as expected, the presence of halides cancels the selectivity of the sulfoxidation reactions entirely.22,25
The vanadium haloperoxidases are named according to their oxidizing ability; e.g. bromoperoxidases
are capable of oxidizing both iodide and bromide, whereas chloroperoxidases can also oxidize chloride.
The vanadium bromoperoxidases originate primarily from seaweeds26 and the vanadium chloroperoxi-
dases are mainly found in terrestrial fungi.27 One fundamental feature shared by this group of enzymes
is their high stability, as the vanadium haloperoxidases were shown to remain fully active in the presence
of organic solvents and high concentrations of aggressive oxidants, in addition to their stability towards
elevated temperatures.28–31
In previous studies it has been demonstrated that vanadium bromoperoxidases are capable of catalyzing
the sulfoxidation of small organic sulfides with very high enantioselectivity.22,23,25 The bromoperoxidase
from the brown seaweed Ascophyllum nodosum produces the (R)-enantiomer of methyl phenyl sulfoxide
(91% ee) under slightly acidic conditions, while the bromoperoxidase from the red seaweeds Corallina
pilulifera produces the (S)-enantiomer of this sulfoxide (55% ee).22 The bromoperoxidase from the red
seaweed Corallina officinalis was observed to convert organic sulfides structurally resembling indenes
to the (S)-enantiomer of the corresponding sulfoxides showing high selectivities (91% ee).23 In addition,
it has been reported that small sulfides, possessing a cis-positioned carboxyl group with respect to the
sulfur atom, are rapidly converted to the corresponding sulfoxide with selectivities exceeding 95% ee in
the presence of vanadium bromoperoxidase from C. officinalis.25 Remarkably, methyl phenyl sulfide and
many derivatives resembling this sulfide are hardly oxidized by this enzyme.23,25
Presumably, the high selectivity of the reaction is due to the direct transfer of oxygen from the
peroxide bound to the vanadium in the active site to the organic sulfide. X-Ray crystallography data have
shown for the vanadium chloroperoxidase from the fungus Curvularia inaequalis that such a peroxo-
enzyme intermediate, with the peroxide coordinated side-on to the vanadium, is formed during halide
oxidation catalysis.32 The sulfide might bind near or in the active site to facilitate the oxygen transfer
from the bound peroxide to the substrate. The presence of a binding site for organic substrates in the
bromoperoxidase from A. nodosum was suggested previously.33
The sulfoxidation activity of vanadium bromoperoxidase from A. nodosum in enantioselective sulfoxi-
dation reactions is described here, and the electronic influence of para-substituents on the aromatic ring
of methyl phenyl sulfide on the conversion and selectivity of the enzymatic oxidation is examined. In
addition, the ability of this enzyme to oxidize a broad range of sulfides of various size and substitution
patterns was probed. It is shown for the first time that the vanadium bromoperoxidase from A. nodosum
mediates the sulfoxidation of racemic non-aromatic cyclic thioethers with high kinetic resolution.

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2. Results and discussion
2.1. Electronic effects
Substrate specificity in the sulfoxidation catalyzed by vanadium bromoperoxidase from the brown
seaweed A. nodosum was studied using several organic sulfides in the presence of hydrogen peroxide.
First we investigated the electronic effects on vanadium bromoperoxidase sulfoxidation activity using
para-substituted phenyl methyl sulfides. The conversion, enantiomeric excess (ee) and configuration of
the resulting sulfoxides are reported in Table 1. The incubation period lasted for 3 days in order to assure
sufficient conversion to be able to analyze the products by NMR. A relatively low enzyme concentration
had to be used, compared to the heme peroxidase mediated sulfoxidations, as the quantity of enzyme
isolated and purified from the seaweed is rather low at present.34,35 Attempts are being made to develop a
recombinant expression system, as has been successfully done for the vanadium chloroperoxidase from
the fungus C. inaequalis.36
Table 1
Sulfoxidation of para-substituted methyl phenyl sulfides by vanadium bromoperoxidase from A.
nodosum at pH 5.3
Evidently, activating substituents on the para-position of the aromatic ring, such as -NH2 or -OCH3,
result in an increase in the selectivity of the sulfoxidation of the phenyl methyl sulfide from 76% ee
for 1d to up to 89% ee for 1a. By contrast, strong electron withdrawing groups, such as -CN 1h and
-NO2 1i have a dramatic effect on the selectivity and conversion of the reaction. Sulfides containing these
substituents are found to be hardly converted and the product, if detected, was found to be racemic.
A similar tendency was found when lactoperoxidase was used in the conversion of these sulfides.37 In
contrast, the results obtained using the heme chloroperoxidase18,19 and horseradish peroxidase38 in these
sulfoxidations show that the enantioselectivity of these enzymes is not influenced by electronic effects.
However, these results should be interpreted with care. When the conversion and enantiomeric excesses
are determined for the sulfoxide produced by heme peroxidases, the yields of the racemic product formed
in the non-enzymatic sulfoxidation reactions are subtracted from the yields of the enzymatic product.
Consequently, the reported enantioselectivities in the sulfoxidations catalyzed by horseradish peroxidase
are artificially high, and cannot be compared with the selectivities found in this and previous studies
on vanadium peroxidase mediated sulfoxidation.22,23,25 In addition, early reports on the sulfoxidation
activity of the heme chloroperoxidase using tert-butyl hydroperoxide show para-substitution dependency
similar to the results obtained in this contribution.14
Table 1 shows that the conversion observed after 3 days of incubation for the various substrates is

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almost similar in the presence or absence of enzyme. The latter non-enzymatic process produces a
racemic mixture. However, the relative contribution of the two processes to the determined conversion
and selectivity of the reaction is difficult to assess. For the sulfoxidation reaction in the presence of
enzyme the initial rate of conversion in the non-enzymatic reaction may closely parallel that of the
enzyme-catalyzed reaction, however as the substrates are consumed, the contribution of the enzymatic
reaction becomes more important.22 Therefore, higher selectivities are obtained when sulfides are used,
which are more readily oxidizable for the enzyme, such as 1a to 1d. In the absence of enzyme, only
the non-enzymatic sulfoxidation reaction consumes the substrates and determines the rate of conversion.
Correction of the determined selectivity by the contribution of the non-enzymatic reaction would lead to
extremely high and unlikely selectivities for the enzyme-catalyzed reaction.
For 1d and 1g it is observed that the conversion of the non-enzymatic reaction exceeds that of
the enzymatic one. It is difficult to explain why a higher conversion is found in the non-enzymatic
sulfoxidation of methyl phenyl sulfide than in the enzymatic reaction. It is possible that hydrogen
peroxide is gradually non-productively consumed in the presence of the vanadium enzyme due to
impurities present in the enzyme preparation, as has been seen before.22 As a consequence of this
hydrogen peroxide consumption the conversion of the sulfide does not reach completion in the enzymatic
reaction.
From Table 1 it can also be inferred that the apparent selectivity of the vanadium bromoperoxidase
mediated sulfoxidation of methyl phenyl sulfide 1d is only moderate, 76% ee, compared to the results
of previous studies where the enzyme converted the sulfide with 91% ee.22 The low selectivity of the
reaction is attributed to a larger contribution of the non-enzymatic sulfoxidation reaction under the reac-
tion conditions used in the present study. Previous studies22,23,25 show that vanadium bromoperoxidases
exhibit high enantioselectivity, however the enantioselectivity and versatility of the heme chloroperoxi-
dase from C. fumago in the biotransformation of sulfides is still better.18,19 We believe that the difference
in enantioselectivity between the two enzymes is due to the low turnover frequency, which is only 1
min−1 for the vanadium bromoperoxidase21 and at least 103 min−1 for heme chloroperoxidase.19,20,39 As
a result of the long incubation times required to convert the sulfide into the sulfoxide the non-enzymatic
reaction has a relatively large contribution and results, as a consequence, in a decreased selectivity of
the vanadium bromoperoxidase formed methyl phenyl sulfoxide. Other heme peroxidases, including
horseradish peroxidase and lactoperoxidase, are observed to catalyze the conversion of methyl phenyl
sulfide with lower selectivity, at best 80% ee with lactoperoxidase.21 The lower selectivity is also due
to oxidative inactivation which also greatly suppresses the turnover frequency during catalysis.21,37,40
Further investigations are being conducted in order to optimize the sulfoxidation activity of the vanadium
bromoperoxidase from the brown seaweed.
2.2. Substrate screening
Several substituted methyl phenyl sulfides, benzyl methyl sulfides and non-aromatic organic sulfides
were used as substrates in the sulfoxidation reaction catalyzed by vanadium bromoperoxidase in order
to investigate the scope of the vanadium enzyme from the brown seaweed. Some results are presented
in Table 2. The reactions were conducted at room temperature over a long reaction time (3 days) due to
low enzyme availability in order to ensure sufficient conversion of all substrates for 1H NMR analysis.
Hydrogen peroxide was added in five sequential steps of 12 h apart during the reaction to decrease the
non-stereoselective contribution of the non-enzymatic reaction and to obtain higher selectivity.
From Table 2 it can be inferred that substitution at the para-position of the aromatic ring is important
for controlling selectivity for both the dimethyl substituted phenyl methyl sulfides and the substituted

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Table 2
Sulfoxidation of several organic sulfides catalyzed by vanadium bromoperoxidase from A. nodosum
at pH 5.3
benzyl methyl sulfides. para-Substituted dimethyl phenyl methyl sulfides 2 and 3 were observed to
be converted with nearly similar or higher selectivity [62% ee and 81% ee (R), respectively], than the
unsubstituted phenyl methyl sulfide 1d [76% ee (R)] under identical reaction conditions.
Unsubstituted benzyl methyl sulfoxide 7 is produced by the vanadium enzyme with very low enan-
tioselectivity [6% ee (R)]. However, introducing a substituent on the aromatic ring results in a more
selective conversion. Thus, introduction of activating groups such as -OCH3 and -CH3 at the para-
position of benzyl methyl sulfide slightly increases the selectivity of the sulfoxidation reaction [16%
ee and 27% ee (R), respectively], and the presence of a carboxyl group elevates the selectivity to 60%
ee (R). No trend can be detected for the benzyl methyl sulfides concerning the electronic influence of
the para-substituents. An amino substituent at the ortho- or para-position, 11a and 11c, hardly increases
the selectivity of the conversion compared with the unsubstituted substrate, but substitution at the meta-
position results in a dramatic increase in stereoselectivity [66% ee (R)]. The presence of a nitro group,

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a strong electron-withdrawing group, on the aromatic ring was found to cancel the selectivity of the
reaction completely (results not shown).
The majority of aromatic sulfides where the distance between the aromatic group and the sulfur in
aromatic sulfides is greater than two carbon atoms or those bearing an alkyl group other than -CH3 could
not be converted by the vanadium bromoperoxidase, indicating that sulfides exceeding benzyl methyl
sulfides in size are not converted by the enzyme. In contrast, the vanadium bromoperoxidase is capable
of catalyzing larger sulfides, which are sterically more constrained with respect to the enzyme than 1d,
as the catalyzed sulfoxidation of the naphthalene derivative 12 resulted in a small amount (6%) of the
corresponding sulfoxide with 25% ee (R).
The enzyme also selectively oxidizes small cyclic non-aromatic sulfides, bearing a cyclohexyl or a
cyclopentyl group (13), to the corresponding sulfoxides with enantioselectivities ranging from approxi-
mately 10% to 27% ee (R). With the exception of cyclopentylmethyl methyl sulfide (conversion yields
39% of sulfone in both the catalyzed and non-enzymatic reaction) all the organic sulfides studied were
only oxidized to the corresponding sulfoxide.
In previous studies24 it has been shown that a carboxyl group at the ortho-position of several aromatic
and at the cis-position, with respect to the sulfur atom, of non-aromatic sulfides is a prerequisite
for selectivity in the biotransformations mediated by vanadium bromoperoxidase from C. officinalis.
However, the vanadium enzyme from A. nodosum does not show such a strong preference. Moreover,
substituents at the ortho-position of aromatic sulfides do not greatly affect the selectivity of the reaction
(Table 2). We believe that for the latter, vanadium bromoperoxidase substituents at the meta-position are
more relevant for selectivity as can be seen from the results of the sulfoxidation of sulfide 3 and 11b.
However, more experiments with meta-substituted aromatic sulfides have to be performed to prove this
assumption.
Evidently, our data suggest that the vanadium bromoperoxidase from the brown seaweed exhibits
a more versatile substrate requirement than the enzyme from the red seaweed C. officinalis, as it
is not restricted to carboxyl substituted sulfides. However, the selectivities of the transformations
are still relatively low due to the reaction conditions. Optimization of the sulfoxidation reactions
by continuous addition of low concentrations of hydrogen peroxide,21,41 elevation of the reaction
temperature and reduction of the incubation period by increasing the enzyme concentration, may result
in higher enantioselectivities and conversions.
2.3. Kinetic resolution
In the oxidation of racemic sulfides 15 and 16, containing either a five-member or six-member cyclic
ether group, respectively, by the bromoperoxidase from A. nodosum to the corresponding sulfoxide, it
was observed that the enzyme exhibits a clear preference towards one of the stereoisomers of 15 and 16.
The results are depicted in Fig. 1. As two separate selective processes occur simultaneously in the sulfo-
xidation catalyzed by the vanadium bromoperoxidase, both kinetic resolution and asymmetric catalysis,
it is difficult to define the selectivity of the reaction. Therefore the excess of one isomer over another is
defined in terms of ratios. In the sulfoxidation of racemic sulfide 15 by vanadium bromoperoxidase it was
found that the stereoisomers of this sulfide were converted with a ratio of approximately 98 to 2, which
means that predominantly one stereoisomer is converted to the corresponding sulfoxide. A less distinct
preference was observed in the enzymatic sulfoxidation of racemic sulfide 16, as a ratio of approximately
10 to 1 was found for the conversion of the two stereoisomers of this sulfide. From the results presented
in Fig. 1, E values of approximately 10 and 30 (for the conversion of 16 and 15, respectively) can be

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4569
calculated.44,45 At present the configuration favored by the vanadium bromoperoxidase has not been
determined.
Fig. 1. Sulfoxidation of racemic non-aromatic cyclic thioethers by vanadium bromoperoxidase from A. nodosum at pH 5.3. The
values in parentheses are the conversions obtained in the absence of enzyme
The sulfoxides produced from the racemic sulfides 15 and 16 consist of two pairs of diastereoisomers
(in CDCl3, 300 MHz, 15: δ (ppm)=2.66 and 2.70 (3H, CH3) and 16: δ (ppm)=2.61 and 2.65 (3H, CH3)),
which can be separately observed by NMR analysis. Upon addition of (S)-(+)-α-methoxyphenyl acetic
acid43 all four isomers can be observed (in CDCl3, 300 MHz, 15: δ (ppm)=2.667, 2.684, 2.693 and 2.703
(3H, CH3) and 16: δ (ppm)=2.630, 2.650, 2.680 and 2.692 (3H, CH3)). For comparison the sulfoxidation
reactions were also conducted in the absence of enzyme yielding racemic mixtures of the products, as
expected. NMR analysis of the products formed in the enzymatic and non-enzymatic reaction did not
indicate overoxidation to sulfone. The ratio between the major diastereoisomers of the sulfoxide formed
in the conversion of racemic sulfide 15 was found to be 5 to 1. Presumably, the major product formed
in this enzymatic conversion is the (R)-enantiomer of the sulfoxide corresponding to the favored isomer
of racemic sulfide 15 (Fig. 1). The ratio between the products formed in the enzymatic sulfoxidation of
the less favored isomer of 15 could not be determined. Similar results were obtained for the enzyme-
catalyzed sulfoxidation of 16 yielding a slightly lower selectivity for the formation of the major product,
as the ratio between the major diastereoisomers of the sulfoxide produced was determined to be 5 to 2.
The absolute configurations of the major products formed in the enzymatic sulfoxidation of sulfides 15
and 16 are probably identical, however the absolute configurations still have to be elucidated.
Nevertheless, these results establish that vanadium peroxidases not only catalyze sulfoxidation reac-
tions through asymmetric catalysis starting with a prochiral substrate as has been shown before,22,23,25
but also distinguish the most suitable substrate from a mixture of isomers in the sulfoxidation reaction.
Therefore these vanadium enzymes are also capable of mediating sulfoxidation reactions based upon
kinetic resolution with high selectivity. This feature of the vanadium peroxidases has not been reported
before.
Surprisingly, the VBPO from A. nodosum is the only asymmetric vanadium-dependent catalyst
converting organic sulfides bearing a phenyl group to the (R)-enantiomer of the corresponding sulfoxide
at present. Several chiral Schiff-base ligated vanadium(V) peroxo-complexes have been shown to mediate
the formation of the (S)-sulfoxides from (substituted) methyl phenyl sulfide with moderate selectivities of
up to 78% ee.46,47 In addition, the VBPOs isolated from the red seaweeds C. pilulifera and C. officinalis
were observed to catalyze the production of the (S)-sulfoxide from methyl phenyl sulfide derivatives.22,25

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H. B. ten Brink et al. / Tetrahedron: Asymmetry 10 (1999) 4563–4572
Unfortunately it is not yet possible to compare the active sites of the VBPOs, because there are no X-
ray structures available of these enzymes, although preliminary X-ray analysis has been reported several
years ago for both the VBPO from A. nodosum and C. officinalis.48,49 We believe, however, that the
environment of the active site of these enzymes will differ substantially. In spite of the primary sequence
analogy between the vanadium haloperoxidases in the active site,36 the presence or absence of a particular
amino acid in the active site may determine the sulfoxidation activity of the enzyme. This has been shown
for horseradish peroxidase, because mutating phenylalanine 41 to a leucine was observed to remarkably
increase both the rate and enantioselectivity of the reaction, while opposite effects were obtained by
mutating to threonine.50 The development of a recombinant expression system for the VBPO from A.
nodosum will allow us to produce various vanadium enzymes by site-directed mutagenesis in order to
study the involvement of the active site amino acids on the sulfoxidation activity and eventually create
new enantioselective enzymes.
3. Experimental
3.1. General procedures
The following sulfides were purchased from Sigma Aldrich; 4-aminophenyl methyl sulfide 1a, methyl
p-methoxy phenyl sulfide 1b, methyl p-tolyl sulfide 1c and methyl phenyl sulfide 1d. Hydrogen peroxide
was purchased from Merck and the concentration was determined to be 9.2 M by spectrophotometric
analysis at 240 nm. The synthesis and characterization of sulfides 1e–1i and 2–14 and the corresponding
sulfoxides have been performed at Brock University in the group of Prof. H. Holland and have been
reported previously.7,8,51–53 Substrates 15 and 16 were prepared by reaction of the corresponding halides
with sodium thiomethylate as previously described.7,51,52 NMR spectra were recorded at 300 MHz
(routine 1H) with a Bruker AC300 using CDCl3 as solvent. The vanadium bromoperoxidase was isolated
from the brown seaweed A. nodosum and purified as described before.21,34,35
3.2. Enzymatic sulfoxidation
The sulfoxidation of different organic sulfides was carried out using the following reaction conditions:
in 10 ml of 100 mM sodium citrate/NaOH buffer, pH 5.3, the organic sulfide (2.5 mM) was incubated
with hydrogen peroxide (final concentration 2.5 mM) and vanadium bromoperoxidase from A. nodosum
(1 µM) for 3 days at room temperature. The sulfides were added dissolved in methanol (100 mM stock
solution) and therefore the reaction mixtures contained 2.5% methanol, however at this concentration this
does not influence the sulfoxidation reaction. The hydrogen peroxide was added in five equal sequential
steps (of 0.5 mM) with approximately 12 h between each addition during the reaction in order to minimize
the contribution of the non-enzymatic reaction between the sulfide and hydrogen peroxide. The non-
enzymatic reaction was carried out under the same conditions.
After three days the samples were quenched with 2 ml of a saturated sodium sulfite solution and
extracted with 2×25 ml of dichloromethane. The organic fractions were pooled, evaporated and dissolved
in approximately 1 ml of CDCl3. The conversion and enantiomeric excess of the enzymatic and non-
enzymatic reactions were determined by NMR analysis at 300 MHz. The conversion of the reaction was
calculated from the integrals of the remaining sulfide and produced sulfoxide. Enantiomeric ratios were
determined in the presence of either (S)-(+)-α-methoxyphenylacetic acid (MPAA reagent) or (R)-(−)-N-
(3,5-dinitrobenzoyl)-α-methylbenzylamine (Kagan reagent).42,43

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Acknowledgements
This work was supported by the BioCentrum and the Innovation Oriented Research Programs Catalysis
(IOP Catalysis). We also received financial support from the Council for Chemical Sciences (CW) of the
Netherlands Organization for Scientific Research (NOW) and The Netherlands Technology Foundation
(STW). The work in Canada was supported by the Natural Sciences & Engineering Research Council
of Canada. This work has been performed under the auspices of NIOK, The Netherlands Institute for
Catalysis Research, Laboratory report no. 99-1-01.
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1
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