Asymmetric Sulfoxidation Catalyzed by a Vanadium-Containing Bromoperoxidase

Article abstract of DOI:10.1021/jo9712456

A vanadium-containing bromoperoxidase (VBrPO) from the alga Corallina officinalis has been shown to catalyze the stereoselective oxidation of some aromatic bicyclic sulfides to the corresponding (S)-sulfoxides in high (up to 91%) ee. Hydrogen peroxide was found to have a large effect on the catalyzed reaction, most likely due to an inhibition of VBrPO. High optical and chemical yields were found to be favored by a continuous slow addition of hydrogen peroxide to keep a low excess. The reaction gives no overoxidation to sulfone, and its stereochemistry is the opposite as compared to that previously found with the heme-containing chloroperoxidase (CPO) from Caldariomyces fumago.

Full text of DOI:10.1021/jo9712456

J . Org. Chem. 1997, 62, 8455-8458
8455
Asym m etr ic Su lfoxid a tion Ca ta lyzed by a Va n a d iu m -Con ta in in g
Br om op er oxid a se
Malin Andersson,† Andrew Willetts,‡ and Stig Allenmark*†
Department of Chemistry, University of Go¨teborg, S-41296 Go¨teborg, Sweden, and
Department of Biological Sciences, University of Exeter, Exeter, EX4 4QD, U.K.
Received J uly 9, 1997X
A vanadium-containing bromoperoxidase (VBrPO) from the alga Corallina officinalis has been
shown to catalyze the stereoselective oxidation of some aromatic bicyclic sulfides to the corresponding
(S)-sulfoxides in high (up to 91%) ee. Hydrogen peroxide was found to have a large effect on the
catalyzed reaction, most likely due to an inhibition of VBrPO. High optical and chemical yields
were found to be favored by a continuous slow addition of hydrogen peroxide to keep a low excess.
The reaction gives no overoxidation to sulfone, and its stereochemistry is the opposite as compared
to that previously found with the heme-containing chloroperoxidase (CPO) from Caldariomyces
fumago.
In tr od u ction
monochlorodimedone)8 and hydrobromination,9 by utiliz-
ing hydrogen peroxide as oxygen source. As with heme
haloperoxidases, disproportionation of hydrogen peroxide
to give molecular oxygen has been observed, albeit with
the difference that this only occurs in the presence of
halide ions yielding singlet oxygen and is consequently
not classified as a classical catalase activity.10 Kinetic
investigations have indicated that halogenation and
dioxygen formation catalyzed by VBrPO proceed through
a common intermediate. A bi-bi ping-pong mechanism
preceding this intermediate, with binding of hydrogen
peroxide prior to halide, has been suggested.7
The VBrPOs characterized thus far all resemble each
other in amino acid composition, vanadium content,
isoelectric point, etc.7 Each consists of a characteristic
albeit different number of subunits, which all have a size
of ca. 65 kDa. Maximum activity is achieved with one
vanadium atom per subunit. During isolation the vana-
dium atom/subunit ratio falls to about 0.4, and full
activity has to be restored by dialysis in an orthovanadate
buffer.11 The vanadium site is believed to be a distorted
octahedron with the vanadium atom coordinated by a
single terminal oxygen, two equatorial nitrogens, and a
further three light-atom ligands.7 The vanadium atom
has been shown to be in its highest oxidation state (+5)
both in the resting state of the enzyme and throughout
catalytic turnover. Consequently hydrogen peroxide will
not oxidize the vanadium ion but is rather thought to
coordinate in a bidentate manner.
Biocatalytic methods have been demonstrated to be
excellent alternatives to chemical procedures for a wide
range of asymmetric organic reactions. One example is
the asymmetric oxidation of prochiral sulfides to optically
active sulfoxides, which has been performed by many
different techniques.1 Of the various biocatalysts exam-
ined,2 chloroperoxidase from the fungus Caldariomyces
fumago (CPO)3 has been shown to be one of the more
successful enzymes in terms of the enantioselectivity of
sulfoxidation4,5 as well as epoxidation.6 Chloroperoxidase
belongs to the class of haloperoxidases characterized by
the presence of an iron heme unit in the active site.
Another unique class of haloperoxidases with a vana-
dium-dependent catalytic activity has recently been
discovered. Several vanadium-containing bromoperoxi-
dases (VBrPO) from marine algae have been character-
ized.7 They all catalyze halide-assisted biotransforma-
tions, such as bromination of organic substrates (e.g.
† University of Go¨teborg.
‡ University of Exeter.
X Abstract published in Advance ACS Abstracts, November 1, 1997.
(1) (a) Bolm, C.; Bienewald, F. Angew. Chem., Int. Ed. Engl. 1995,
34, 2640-2642. (b) Brunel, J .-M.; Diter, P.; Duetsch, M.; Kagan, H. B.
J . Org. Chem. 1995, 60, 8086-8088. (c) Palucki, M.; Hanson, P.;
J acobsen, E. N. Tetrahedron Lett. 1992, 33, 7111-7114.
(2) (a) Ble´e, E.; Schuber, F. Biochemistry 1989, 28, 4962-4967. (b)
Katapodis, A. G.; Smith, J r., H. A.; May, W. S. J . Am. Chem. Soc. 1988,
110, 897-899. (c) Colonna, S.; Gaggero, N.; Pasta, P.; Ottolina, G.
Chem. Commun. 1996, 2303-2307. (d) Takata, T.; Yamazaki, M.;
Fujimori, K.; Kim, Y. H.; Iyanagi, T.; Oae, S. Bull. Chem. Soc. J pn.
1983, 56, 2300-2310.
An interesting feature of these enzymes is their
remarkably high stability toward organic solvents, chelat-
ing agents, and elevated temperatures, properties ap-
preciated in a synthetic application.8c
The aim of this work was to explore whether these
VBrPOs are able to catalyze oxidation of sulfides and
further whether any resultant enantioselectivity would
be found, suggesting that the oxidation takes place within
an active site.
(3) Morris, D. R.; Hager, L. P. J . Biol. Chem. 1966, 241, 1763-1768.
(4) (a) Colonna, S.; Gaggero, N.; Manfredi, A.; Casella, L.; Gullotti,
M.; Carrea, G.; Pasta, P. Biochemistry 1990, 29, 10465-10468. (b) Fu,
H.; Kondo, H.; Ichikawa, Y.; Look, G. C.; Wong, C.-H. J . Org. Chem.
1992, 57, 7265-7270. (c) Colonna, S.; Gaggero, N.; Casella, L.; Carrea,
G.; Pasta, P. Tetrahedron: Asymmetry 1992, 3, 95-106.
(5) (a) Allenmark, S. G.; Andersson, M. Tetrahedron: Asymmetry
1996, 7, 1089-1094. (b) Allenmark, S. G.; Andersson, M. Chirality, in
press.
(6) (a) Zaks, A.; Dodds, D. R. J . Am. Chem. Soc. 1995, 117, 10419-
10424. (b) Colonna, S.; Gaggero, N.; Casella, L.; Carrea, G.; Pasta, P.
Tetrahedron: Asymmetry 1993, 4, 1325-1330. (c) Allain, E. J .; Hager,
L. P.; Deng, L.; J acobsen, E. N. J . Am. Chem. Soc. 1993, 115, 4415-
4416.
(7) Butler, A.; Walker, J . V. Chem. Rev. (Washington, D.C.) 1993,
93, 1937-1944.
(8) (a) Wever, R.; Plat, H.; de Boer, E. Biochim. Biophys. Acta 1985,
830, 181-186. (b) Soedjak, H. S.; Butler, A. Biochim. Biophys. Acta
1991, 1079, 1-7. (c) Sheffield, D. J .; Harry, T.; Smith, A. J .; Rogers,
L. J . Phytochemistry 1993, 32, 21-26.
Only halide-assisted oxidations have been observed
thus far, and the VBrPOs have been claimed to be
(9) Coughlin, P.; Roberts, S.; Rush, C.; Willetts, A. Biotechnol. Lett.
1993, 15, 907-912.
(10) (a) Everett, R. R.; Butler, A. Inorg. Chem. 1989, 28, 393-395.
(b) Everett, R. R.; Kanofsky, J . R.; Butler, A. J . Biol. Chem. 1990, 265,
4908-4914.
(11) Yu, H.; Whittaker, J . W. Biochem. Biophys. Res. Commun. 1989,
160, 87-92.
S0022-3263(97)01245-0 CCC: $14.00 © 1997 American Chemical Society

8456 J . Org. Chem., Vol. 62, No. 24, 1997
Andersson et al.
Ta ble 1. Resu lts Obta in ed fr om In itia l Attem p ts to
Asym m etr ica lly Oxid ize Su lfid es by VBr P O a t p H 6.5 a n d
25 °C
Ta ble 2. Resu lts Obta in ed in VBr P O-Ca ta lyzed
Oxid a tion of 2 a t p H 6.5 a n d 25 °C a fter 2 h Rea ction
temp
(°C)
yieldb
(%)
ee
(%)
time
(h)
VBrPO
(units)
yieldb
(%)
ee
(%)
scalea
peroxide
a
substrate
H2O2
c
1
25
25
25
25
40
25
H2O2
H2O2
H2O2
H2O2
11.5 (0.2)
16 (<0.1)
16
87
72
70
72
74
<1
c
1
1
2
2
2
2
2
2
2
2
6
2
2
2
1
5
1
1
5
5
5
A
B
A
A
A
B
C
2.0
-
-
37
39
65
84
87
1/5
2/5
1/5d
1/5
1/5
c
1.7 (1.2)
13 (8.2)
40 (25)
20 (5.9)
31 (4.4)
42 (2.5)
c
16
c
H2O2
74 (22)
6.3 (2.3)
tBuOOHe
a
This denotion of scale is relative to 25 µmol of 2 and 5 units
b
of VBrPO in 3 mL of buffer. Values within brackets correspond
a
In method A 2 equiv of hydrogen peroxide were added at the
beginning of the reaction, whereas in method B the hydrogen
peroxide was added in equal aliquots every 5 min and in method
to yields obtained in the absence of enzyme. c Two equivalents of
hydrogen peroxide were added continuously during the 2 h
d
reaction time. The stirring rate was decreased from 700 to 200
b
C continuous addition was used. Values within brackets cor-
rpm. e Two equivalents of tert-butyl hydrogen peroxide were used,
respond to yield obtained in the absence of enzyme.
and the reaction was carried out for 5 h.
completely inactive in the absence of halide since at-
tempted oxidations of guaiacol and pyrogallol, which are
typical peroxidase substrates, have been reported to be
unsuccessful.12
In the present investigation VBrPO from the red alga
Corallina officinalis was used.11,13 A series of sulfides,
previously studied together with CPO,5 were used as
substrates. Below we describe for the first time the use
of VBrPO as an efficient catalyst for asymmetric sulfoxi-
dation and compare its catalytic and stereoselective
properties with that of CPO.
2 has been shown to be oxidized by CPO to the corre-
sponding (R)-sulfoxide in 99% ee and quantitative yield.5a
In contrast, the (S)-configuration was obtained with
VBrPO. The mode of addition of hydrogen peroxide was
found to be important since both yield and ee increased
when the hydrogen peroxide was added progressively
throughout the reaction. This phenomenon has been
observed previously in CPO-catalyzed sulfoxidation, where
it is caused by a competing catalase activity resulting in
a hydrogen peroxide decomposition into molecular oxygen
and water.
Since 2 shows poor solubility in the aqueous buffer,
the possibility of using different cosolvents was examined.
The VBrPO activity has been shown to be unaffected by
the presence of different cosolvents, e.g. E40% of alco-
hols.13 A reaction mixture containing 20% ethanol,
however, gave a reduced activity in the oxidation of 2.
On the other hand, the use of a minimum volume of
1-propanol to dissolve 2 prior to addition, resulting in a
final concentration of 0.6% in the reaction mixture,
slightly increased the chemical and optical yield. This
modification, together with an automatized, continuous
addition of hydrogen peroxide, resulted in reproducible
results as long as VBrPO from the same preparation was
used. The scale of the reaction was reduced to 1/5 to
decrease the consumption of VBrPO, resulting in an
increased yield and decreased ee, the rate enhancement
most likely being caused by a more efficient mixing,
leading to increased contact between the enzyme and the
suspended sulfide, Table 2.
Resu lts a n d Discu ssion
In itia l Stu d ies. The first attempt to use VBrPO as a
catalyst in asymmetric oxidation of sulfides was carried
out with methyl p-tolyl sulfide (1), one of the most
frequently used substrates in studies of asymmetric
sulfoxidation. The experimental conditions applied were
based on a modification of the method previously used
with CPO. The reaction was carried out at pH 6.5 since
this is the pH-optimum for the bromination of monochlo-
rodimedone.8c The oxidation of 1, however, was not
catalyzed by VBrPO, Table 1.
tert-Butyl hydrogen peroxide was shown to be a poor
oxygen source in VBrPO-catalyzed sulfoxidation, Table
2. Likewise, alkyl hydrogen peroxides have proved to be
inefficient for the VBrPO-catalyzed generation of a
brominating intermediate, whereas different peracids
have been successfully used.8b,12
In flu en ce of p H a n d Tem p er a t u r e. The pH-
dependence of yield and ee in VBrPO-catalyzed oxidation
of 2 was determined. A relatively flat pH-profile was
obtained over the range pH 4-8, which contrasted with
the more narrow pH-profile of halogenation reactions
which have an optimum at pH 6.5.8c For consistency, a
pH of 6.5 was kept throughout this investigation as no
significant improvement would be expected at a different
pH value within the range 5.5-7.0.
Indene has been found to be a good substrate for
VBrPO-catalyzed hydrobromination14 and 2-substituted
indoles have been suggested to bind into an active site
of a vanadium bromoperoxidase.15 The possibility that
VBrPO would be able to catalyze sulfoxidation of 2,3-
dihydrobenzothiophene (2), a sulfide structurally related
to indene and indole, was therefore examined. This
proved to be a successful approach, and the first results
showing VBrPO to be a potent catalyst in asymmetric
oxidation of sulfides were obtained, Table 1. Previously,
A temperature increase to 40 °C resulted in a much
higher yield while the ee was not significantly affected,
Table 2. It seems likely, though, that the large extent
of uncatalyzed oxidation will prevent any improvement
in ee.
(12) Soedjak, H. S.; Butler, A. Biochemistry 1990, 29, 7974-7981.
(13) Rush, C.; Willetts, A.; Davies, G.; Dauter, Z.; Watson, H.;
Littlechild, J . FEBS Lett. 1995, 359, 244-246.
(14) Mullins, G.; Willetts, A., unpublished results.
(15) Tschirret-Guth, R. A.; Butler, A. J . Am. Chem. Soc. 1994, 116,
411-412.

Bromoperoxidase-Catalyzed Asymmetric Sulfoxidation
J . Org. Chem., Vol. 62, No. 24, 1997 8457
F igu r e 3. Yield and ee as a function of the reaction time in
the oxidation of 2. The hydrogen peroxide was added at a rate
corresponding to a total addition of 2 equiv at the end of each
reaction.
Previous investigations have shown that, in contrast
to CPO, disproportionation of hydrogen peroxide to
molecular oxygen and water by VBrPO only occurs in the
presence of halide.10 Prior to our investigation, however,
catalytic turnover has only been observed in the presence
of halide. With a suitable secondary substrate (e.g.
monochlorodimedone) present, the catalyzed bromination
has been found to totally dominate over any oxygen
formation.10b The possibility that VBrPO could promote
dismutation of hydrogen peroxide during catalytic turn-
over with sulfides was therefore studied, but no oxygen
could be detected in the presence of 2. However, by
exchanging the sulfide for an equal concentration of
bromide, significant O2 release was observed. Interest-
ingly, this was inhibited completely by the presence of 2
in the bromide-containing reaction mixture. Together
these results might indicate that the catalysis of sulfoxi-
dation predominates over the formation of a brominating
intermediate, but alternatively could reflect a reaction
between the sulfide and the brominating intermediate.
F igu r e 1. Influence of the enzyme concentration on yield and
ee in the VBrPO-catalyzed oxidation of 2 for 2 h (a) and 3 for
16 h (b), respectively at 25.0 °C.
The lack of oxygen formation in the catalyzed sulfoxi-
dation reaction ruled out the disproportionation of hy-
drogen peroxide to account for the disfavorable effect of
high levels of hydrogen peroxide on the reaction. An
inhibition of vanadium bromoperoxidase catalysis by
hydrogen peroxide has been suggested earlier16 and
should be the most likely cause here as well. The rate
of product formation, illustrated by Figure 3, shows a
distinct sigmoidal curvature which can be explained by
an inhibition by hydrogen peroxide over the shorter
reaction times.
Another remarkable feature of the hydrogen peroxide
influence on VBrPO is demonstrated in Figure 2. The
ee continuously decreases during the progress of the
reaction. The uncatalyzed reaction was found to be
negligible (<0.1%, Table 2) over the same 2 h period and
consequently cannot account for this phenomenon. In-
stead, an inhibition by hydrogen peroxide could account
for the decrease in stereoselectivity, an assumption
supported by the data shown in Figure 3. The prolonged
reaction time would rather decrease the ee due to a
higher extent of uncatalyzed reaction; however, the
opposite is observed. A higher concentration of hydrogen
peroxide will be present when hydrogen peroxide is added
faster over shorter reaction times, thus exacerbating any
inhibitory effects on the enzyme.
F igu r e 2. Development of yield and ee during the oxidation
of 2 with 1 unit of VBrPO. The hydrogen peroxide was added
at a rate corresponding to an addition of 2 equiv after 2 h.
Va r ia tion of th e En zym e/Su bstr a te Ra tio. The
effect of changing the enzyme/substrate ratio on yield and
ee is shown in Figure 1. A yield of 76% and an ee of 91%
was achieved by using 5.6 units of VBrPO, and the profile
in Figure 1 shows that the yield might be further
increased although a relatively large amount of catalyst
would have to be used. The curve shape is as expected
from Michaelis-Menten kinetics.5b The constant rate of
product formation from 2 found during the first 2 h at
low enzyme concentration is shown in Figure 2.
Effects of Hyd r ogen P er oxid e. Hydrogen peroxide
was found to have a significant effect on both yield and
ee in the VBrPO-catalyzed oxidation of 2. This was
clearly demonstrated in an approach designed to obtain
a high ee together with a high yield by prolonging the
reaction time. In this way the hydrogen peroxide can be
added at a slower rate and its concentration kept at a
lower level during the reaction. This proved to be an
excellent method since oxidation of 2 during 16 h resulted
in 98% yield and 90% ee, Figure 3.
(16) (a) Soedjak, H. S.; Walker, J . V.; Butler, A. Biochemistry 1995,
34, 12689-12696. (b) Everett, R. R.; Soedjak, H. S.; Butler, A. J . Biol.
Chem. 1990, 265, 15671-15679.

8458 J . Org. Chem., Vol. 62, No. 24, 1997
Andersson et al.
Ta ble 3. Resu lts Obta in ed a fter 16 h Rea ction in
VBr P O-Ca ta lyzed Asym m etr ic Oxid a tion of Su lfid es a t
p H 6.5 a n d 25 °C
Exmouth, UK. Washed biomass (450 g) was mascerated in 1
L of chilled 50 mM Tris-sulfate buffer, pH 8.0. Following
centrifugation (3000g, 20 min at 4 °C), the protein precipitated
after treatment with 60% ammonium sulfate and was centri-
fuged out of solution (3000g, 20 min at 4 °C) and resuspended
in minimum volume of 50 mM Tris-sulfate buffer, pH 8.0.
Following overnight dialysis at 4 °C against 2 L of the same
buffer containing 1 mM sodium orthovanadate, the sample was
centrifuged (10000g, 20 min at 4 °C) and any precipitated
material discarded. The dialysate was frozen at -15 °C and
lyophilized until completely desiccated. No significant loss in
enzyme activity (total units extracted from source material)
occurred throughout these procedures, or on subsequent
storage of the freeze-dried preparation at 4 °C for periods up
to 6 months.
The purified enzyme was dissolved in 50 mM Tris-sulfate
buffer, pH 8.0 and stored at 4 °C. The enzyme stock solutions
had a specific activity in the range of 135-175 units/mL,
determined spectrophotometrically at 25 °C by the monochlo-
rodimedone assay using the extinction coefficient of monochlo-
rodimedone of 19.9 mM-1 cm-1 at 290 nm.13 A 50 mM
phosphate buffer, pH 6.0 was used.
En zym a tic Oxid a tion . The initial oxidation reactions
were performed by mixing sulfide (25 µmol) and 5 units of
VBrPO (stock solution) in 50 mM phosphate buffer, pH 6.5.
The mixture was stirred and thermostated at 25.0 °C. The
hydrogen peroxide (50 µmol) was dissolved in the buffer used
(0.160 M) and added either as one aliquot to initiate the
reaction, stepwise as a series of equal small portions every 5
min, or continuously using an auto injector. The total volume
of the reaction mixture was 3 mL. The reaction was quenched
after the requisite reaction time (2 h) with 1 mL of a saturated
sodium sulfite solution and extracted with 3 × 2 mL of
dichloromethane. The organic phase was dried (MgSO4),
filtered, and then analyzed by enantioselective gas chroma-
tography.5
All reactions, except for those referred to in Table 1, were
carried out with 0.5-0.6% of 1-propanol.
The buffers used for the pH-dependence study were either
50 mM phosphate buffer (pH 6.5-8.0) or 50 mM citrate-
phosphate buffer (pH 4.0-6.3).
By an overall volume reduction the reaction was scaled
down to 1/5 and extraction by 3 × 1 mL dichloromethane was
used in the workup procedure. Reactions referred to in Table
3 and all figures were carried out at this reduced scale.
In the experiment with tert-butyl hydrogen peroxide, 10
µmol was added as a single aliquot to the reaction mixture.
The reaction represented in Figure 2 was studied at a rate
of hydrogen peroxide addition of 1 equiv/h, whereas at each
reaction time given in Figure 3, 2 equiv has been added.
An estimation of the background oxidation in the absence
of enzyme under the same reaction conditions was performed
in most cases.
a
VBrPO
(units)
H2O2
yieldb
(%)
ee
(%)
abs
config
substrate
(equiv)
1
2
2
3
3
3
4
5
6
1
1
1
1
1
6
1
1
1
2
2
1.1
2
1.1
1.1
1.1
1.1
1.1
6 (6)
99
0
89
90
49
63
91
-
-
S
S
S
S
99 (38)
25 (12)
19 (6.1)
84
1.3 (0)
25 (9.0)
99 (58)
S
-
R
-
38
-
a
The continuous addition of hydrogen peroxide was completed
b
after 16 h reaction time. Values within brackets correspond to
yields obtained in the absence of enzyme.
Su bstr a te Recogn ition . A series of sulfides were
oxidized, using the optimized experimental conditions,
to give an initial view of the capacity of VBrPO as an
asymmetric catalyst, Table 3. Again compound 1 ap-
peared as a nonsubstrate as the uncatalyzed reaction
completely dominated. The steric tolerance of the active
site was explored by increasing the size of the sulfide
from the five-membered ring in 2 to a six-membered ring,
as in thiochroman 3. However, the oxidation of 3 was
less effectively catalyzed, and some further experiments
to improve the outcome of the reaction were carried out.
Decreasing the excess of hydrogen peroxide reduced the
contribution from the uncatalyzed reaction leading to a
decreased yield and an increased ee. By increasing the
VBrPO/substrate ratio both yield and ee were increased
(Table 3 and Figure 1b).
The oxidation of thiochroman-4-one (4) was not cata-
lyzed by VBrPO (Table 3) although the reason for this
nonsubstrate behavior remains to be elucidated.
1,3-Benzoxathiole (5), a structural analogue of 2, was
oxidized by VBrPO with the same stereopreference shown
for 2 and 3 (Table 3). The designation of (R)-configura-
tion is only a consequence of the Cahn-Ingold-Prelog
nomenclature. Although no further attempts to improve
the yield and ee have been performed, this initial result
is of interest since 5 has been shown to act as a
competitive inhibitor of CPO on attempted oxidation.5b
1,3-Dihydrobenzo[c]thiophene (6), the symmetric ana-
logue of 2, was oxidized in quantitative yield. This
indicates that further substrate variations may be toler-
ated by the enzyme.
Mea su r em en t of Oxygen Evolu tion . The Clark-elec-
trode was calibrated using dithionite-containing water (zero)
and air-saturated water (0.247 mM O2).16a The reaction buffer
was sparged with nitrogen gas prior to use to minimize the
O2 content, and the background rate of O2 formation was
measured and compensated for. The assay was initiated by
addition of hydrogen peroxide in one aliquot. The total volume
was 1.2 mL and consisted of 8 mM sulfide, 0.33 units of
VBrPO, and 8 mM hydrogen peroxide in 50 mM phosphate
buffer, pH 6.5. Corresponding reactions were carried out with
9 mM KBr with or without sulfide present.
Exp er im en ta l Section
Gen er a l P r oced u r es. The continuous addition of hydro-
gen peroxide was carried out by use of a syringe pump (Sage
Instruments Model 352; 100 µL or 250 µL syringes). Oxygen
evolution was measured by a Clark-electrode linked to an
oxygraph and O2-electrode control box (Hansa Tech Instru-
ments). Methyl p-tolyl sulfide and the corresponding (R)- and
(S)-sulfoxides were obtained from Aldrich. The synthesis and
characterization of 2-6 and the corresponding sulfoxides have
been reported previously.5 The concentration of hydrogen
peroxide in the buffer solutions was determined spectropho-
tometrically at 240 nm (ꢀ ) 43.6 M-1 cm-1).8c For further
details regarding instrumentation and general procedures, see
refs 5a,b.
Ack n ow led gm en t. This work was supported by a
grant (K-AA/KU 02508-321) from the Swedish Natural
Science Research Council. Thanks are also due to Mrs.
Gillian Mullins for providing the samples of VBrPO.
En zym e P r ep a r a tion a n d Activity Mea su r em en t. The
alga C. officinalis was collected from the intertidal zone at
J O9712456