Chloroperoxidase-catalyzed oxidation of methionine derivatives

Article abstract of DOI:10.1139/v02-025

Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of L- and D-methionine and L-ethionine by chloroperoxidase-hydrogen peroxide resulted in oxidation at sulfur to produce the (R_S) sulfoxide in moderate to high diastereomeric excess.

Full text of DOI:10.1139/v02-025

633
Chloroperoxidase-catalyzed oxidation of
methionine derivatives
Herbert L. Holland, Frances M. Brown, Damian Lozada, Benjamin Mayne,
W. Rick Szerminski, and Aaron J. van Vliet
Abstract: Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of L- and D-methionine and L-ethionine by
chloroperoxidase–hydrogen peroxide resulted in oxidation at sulfur to produce the (RS) sulfoxide in moderate to high
diastereomeric excess. The (RS) sulfoxide of methionine was also obtained in moderate to high diastereomeric excess
from (±)SO-N-methoxycarbonyl-L-methionine methyl ester sulfoxide by ester hydrolysis using -chymotrypsin,
Aspergillus sp. protease or subtilisin Carlsberg.
Key words: amino acid oxidation, biocatalysis, biotransformation, chloroperoxidase, enzyme catalysis, lipase, sulfoxidation.
Résumé : Le traitement de dérivés N-méthoxycarbonyle C-carboxylates des L- et D-méthionines et de la L-éthionine par
de la chloroperoxydase et du peroxyde d’hydrogène conduit à l’oxydation au niveau du soufre et à la formation de
sulfoxydes (RS), avec des excès diastéréomériques qui vont de modérés à élevés. Le (RS) sulfoxyde de la méthionine a
aussi été obtenu avec un excès diastéréomérique élevé à partir de l’ester méthylique du de sulfoxyde (±)SO-N-méthoxy-
carbonyl-L-méthionine, par hydrolyse de l’ester à l’aide d’ -chymotrypsine, de protéase d’Aspergillus sp. ou de subti-
lisine Carlsberg.
Mots clés : oxydation d’acide aminé, biocatalyse, biotransformation, chloroperoxydase, catalyse enzymatique, lipase,
sulfoxydation.
[Traduit par la Rédaction] Holland et al. 639
S
CO2H
NH2
S
CO2H
NH2
CH3
CO2H
NH2
C2H5
C H3
S
Introduction
Sulfoxides of the sulfur-containing amino acids possess
various biological properties. Oxidation of methionine resi-
dues in proteins is the target of continued interest, and can
result in structural and functional changes in protein (1) and
(or) cellular oxidative-stress properties (2). The reversible
oxidation of methionine (1) can also be involved in other bi-
ological functions such as the modulation of potassium-
channel activity (3) and the oxidation of high- density lipo-
proteins (4, 5). It is also implicated in ageing processes by
the effect of methionine sulfoxidation on the activity of the
human keratinocyte growth factor (6) and on the immuno-
peptide thymosin -4 (7); the in vivo oxidation of
methionine to the sulfoxide is an age-dependent process in
humans (8). The carcinogenic properties of ethionine (2)
have been linked to its in vivo oxidation to sulfoxide (9),
while S-methylcysteine sulfoxide (3-sulfoxide) is responsi-
ble for the antibacterial action of cabbage (10) and possesses
the ability to lower serum-cholesterol levels (11, 12).
1
2
3
With the exception of L-methionine sulfoxide isolated
from the blowfly Phormia regina (13), and S-methyl-L-
cysteine sulfoxide from cabbage (14), both of which have
the (SS) configuration, no data are available on the configu-
ration at sulfur in naturally occurring amino acid sulfoxides.
The stereoselective chemical oxidation at sulfur of a thia-
amino acid has not been reported, and only recently have
other chemical routes for the preparation of
diastereomerically defined amino acid sulfoxides been sys-
tematically investigated (14). The value of biocatalytic oxi-
dation for the stereoselective oxidation of sulfides to
sulfoxides, however, is well established (15, 16), and was
used for the oxidation of methionine by Penicillium citreo-
viride, but no stereochemical details were reported (17).
Fungal biocatalysts oxidize various prochiral sulfides to
chiral sulfoxides. The use of Beauveria bassiana ATCC
7159 and B. caledonica ATCC 64970 for the stereoselective
oxidation of N-phthaloyl-methionines and -ethionines to the
(SS) methionine and ethionine sulfoxides has recently been
reported (18, 19). The application of isolated enzymes for
chiral sulfoxidation is less common, but chloroperoxidase
(CPO) from Caldariomyces fumago has been systematically
used for the oxidation of various sulfides (16, 20–22).
Cyclohexanone monooxygenase (CMO) from Acinetobacter
species has also been employed for moderate diastereomeric
sulfoxidation of S-allylcysteine (alliin) (23).
Received 23 November 2001. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 17 April 2002.
This paper is dedicated to Professor J. Bryan Jones, whose
introductory work, continued research, and present-day
creations are all fundamental areas of biocatalysis.
H.L. Holland,1 F.M. Brown, D. Lozada, B. Mayne, W.R.
Szeminski, and A.J. van Vliet. Department of Chemistry,
Brock University, St. Catharines, ON L2S 3A1, Canada.
1Corresponding author (e-mail holland@chemiris.labs.brocku.ca).
Can. J. Chem. 80: 633–639 (2002)
DOI: 10.1139/V02-025
© 2002 NRC Canada

634
Can. J. Chem. Vol. 80, 2002
Table 1. CPO-catalyzed oxidations of L-methionine derivatives.
Table 2. CPO-catalyzed oxidations of D-methionine derivatives.
O
O
S
CO2R
S
CO2R
S
CO2R
NHR'
S
CO2R
NHR'
CH3
CH3
CH3
CH3
NHR'
Sulfoxide
NHR'
Sulfoxide
Conversion
(%)
de
(%)
Conversion
(%)
de
(%)
R
R
H
stereochemistry
R
R
stereochemistry
Ac
MOC
Phthaloyl
Ac
MOC
ClAc
CHO
t-Boc
MOC
MOC
MOC
MOC
0
2
0
—
—
—
72
82
12
30
72
72
84
48
60
—
—
—
R
R
R
R
R
R
R
CH3
MOC
t-Boc
MOC
MOC
MOC
MOC
42
6
22
28
19
10
50
—
50
46
24
0
R
—
R
R
R
C2H5
CH3
20
84
85
25
10
60
72
60
50
n-C3H7
n-C4H9
n-C5H11
—
Table 3. CPO-catalyzed oxidations of L-and D-ethionine derivatives.
C2H5
O
n-C3H7
n-C4H9
n-C5H11
R
R
S
CO2R
S
CO2R
C2H5
C2H5
C-2
NHMOC
NHMOC
Conversion
(%)
de
(%)
Sulfoxide
With the latter exception, the present paper describes the
first use of isolated enzymes, particularly the oxidizing spe-
cies chloroperoxidase–hydrogen peroxide, for the stereo-
selective oxidation at sulfur in methionine and ethionine
derivatives.
configuration
R
stereochemistry
L (S)
L (S)
D (R)
CH3
C2H5
CH3
30
40
8
91
80
0
R
R
—
Results and discussion
consistent excess of the downfield 13C NMR signals in the
stereochemically dependent pairs.
Table 3 illustrates that only the L-ethionine derivatives
were effective substrates for CPO-catalyzed oxidation at sul-
fur. The configurations at sulfur were determined as de-
scribed above, i.e., using 13C NMR spectral analysis of
Table 1 summarizes the optimum use of substrate-
protecting groups for the chloroperoxidase-catalyzed oxida-
tion of L-methionine derivatives, where maximum yield and
diastereomeric excesses were obtained with the N-
methoxycarbonyl (N-MOC) C-carboxylate methyl ester de-
rivatives.
carbons to the sulfoxide sulfur (ca.
24 (C-3) and 51
The configurations at sulfur of the sulfoxides listed in Ta-
ble 1 were obtained by analysis of their 13C NMR spectra.
Although the 1H NMR spectra of the two diastereomers
(SSSC) and (RSSC) were essentially identical, the signals in
13C NMR spectra from the and carbons to the sulfoxide
sulfur were separable in methanol. Comparison with 13C
NMR spectra from diastereomerically pure L-methionine
sulfoxide standards with similar protection groups (19), were
then used to assign the configurations obtained by the CPO-
catalyzed oxidations. The sulfoxides listed in Table 1 were
predominantly (RSSC) configurations, and illustrate a consis-
tent excess of the upfield-signal intensity in the stereo-
chemically dependent pairs of signals at ca. 24 (C-3), 37
(S(O)-CH3), and 50 (C-4). Confirmation that the diastereo-
meric pairs of all the sulfoxide products (possessing both
(RSSC) and (SSSC) configurations) exhibited base-line peak
separation in the 13C NMR spectra was obtained from the
13C NMR spectra of such products obtained using hydrogen
peroxide as the oxidation reagent for the N-MOC ester de-
rivatives.
(SCH2CH3)), and by comparison with the 13C NMR spectra
of stereochemically known samples (19). The predominant
signal from the (RSSC) configurations of both diastereo-
merically enriched sulfoxides occurs as the upfield compo-
nent of the stereochemically dependent pair of signals.
The formation of diastereomerically enriched sulfoxides
by stereoselective hydrolysis of the methyl ester from dia-
stereomeric N-MOC-L-methionine sulfoxides (1:1) is sum-
marized in Table 4.
Of the hydrolytic enzymes that were successful in the
stereoselective hydrolysis of this diastereomeric pair, sub-
tilisin Carlsberg was the most efficient and gave both the
acid and the unreacted ester products in moderate yield and
high diastereomeric excess. Again, configuration at sulfur of
both products was determined by 13C NMR spectral analysis
of the and carbons to the sulfoxide sulfur (ca. 24 (C-3),
37 (S(O)-CH3), and 50 (C-4)), with (RSSC) configurations of
diastereomerically enriched sulfoxides occurring as the
upfield component of the stereochemically dependent signal
pair and (SSSC) configurations with the predominant peak as
the downfield component.
The results obtained from CPO-catalyzed oxidation of D-
methionine derivatives are listed in Table 2. The configura-
tion at sulfur was again determined to be (RS) by 13C NMR
spectral analysis of the and carbons to the sulfoxide sul-
fur (ca. 24 (C-3), 37 (S(O)-CH3), and 50 (C-4)). The pre-
dominant (RSRC) configurations of the products illustrate a
The CPO-catalyzed oxidations of a series of methionine-
containing dipeptides are summarized in Table 5. Although
the enzyme was capable of the oxidation of several of these
substrates in acceptable yields, only one sulfoxide, from the
© 2002 NRC Canada

Holland et al.
635
Table 4. Stereoselective hydrolysis of N-MOC-L-methionine methyl ester sulfoxides.
O
S
O
S
O
S
CO2CH3
NHMOC
CO2H
CO2CH3
NHMOC
CH3
CH3
CH3
+
NHMOC
O
S
O
S
CO2CH3
NHMOC
CO2H
NHMOC
CH3
CH3
Time
(min)
Yield
(%)
de
(%)
Sulfoxide
stereochemistry
Yield
(%)
de
(%)
Sulfoxide
stereochemistry
Enzyme
-Chymotrypsin
Aspergillus sp. protease
Subtilisin Carlsberg
Aspergillus lipase
10
5
15
10
42
39
41
25
82
40
90
0
R
R
R
—
35
46
38
58
80
28
86
0
S
S
S
—
chemical shifts are reported in ppm ( ) and the signals
quoted as s (singlet), d (doublet), t (triplet), q (quartet), or m
(multiplet). The 13C NMR spectra were recorded at 75 MHz
on the same spectrometer in CDCl3, CD3OD, or D2O.
Diastereomeric excess (de) was determined by 13C NMR
analysis of signals and to sulfur. Mass spectra were ob-
tained using a Kratos 1S spectrometer. Optical rotations
were recorded at ambient temperature in the stated solvent
using a Rudolph Autopol 3 polarimeter. TLC was performed
on Merck silica gel F254 plates, 0.2 mm, and column chro-
matography used Merck silica gel 9385, 230–400 mesh.
Chloroperoxidase from Caldariomyces fumago was ob-
tained from the Sigma Chemical Co. (catalogue no. C-0278).
-Chymotrypsin, Aspergillus sp. protease, subtilisin
Carlsberg, and Aspergillus lipase were obtained from Altus
Biologics Inc.
Table 5. CPO-catalyzed oxidations of L-methionine-containing
protected dipeptides
N-MOC-Me-ester
dipeptide
Yield
(%)
de
(%)
Sulfoxide
stereochemistry
MET-GLY
MET-ALA
MET-LEU
MET-PRO
MET-PHE
GLY-MET
ALA-MET
LEU-MET
PRO-MET
PHE-MET
90
80
90
17
<5
95
90
16
33
<5
0
0
0
0
0
0
98
0
5
0
—
—
—
—
—
—
R
—
—
—
methyl ester of N-MOC, ALA-MET, was produced in a
stereochemically acceptable form.
Preparation of substrates
N-Protection
In the absence of a stereochemical standard for this prod-
uct, the assignment of the configuration at sulfur was again
based on 13C NMR spectral analysis of the and carbons
to the sulfoxide sulfur (ca. 24 (C-3), 37 (S(O)-CH3), and
50 (C-4)), with the assumption that the (RSSC) configuration
of N-MOC-ALA-MET methyl ester sulfoxide presumably
resulted in signal intensities consistent with the standard L-
methionine products. Nevertheless, the description of the
methionine sulfoxide configuration of the dipeptide product
as (RSSC) must be considered tentative.
The removal of both N-MOC- and methyl ester protecting
groups from the diastereomerically enriched L-methionine,
D-methionine, and L-ethionine sulfoxide enzyme-catalyzed
products was performed in moderate yield by routine hydro-
lysis with barium hydroxide, generating the appropriate free
amino-acid sulfoxides.
N-t-Boc-L- and D-methionines, N-acetyl-L-methionine, and
N-formyl-L-methionine are commercial samples. N-chloro-
acetyl-L-methionine (24) and N-phthaloyl-L-methionine (19)
were prepared from the amino acids as previously described.
N-MOC-L- and D-methionines, N-MOC-L- and -D- ethionines,
and N-MOC-dipeptides were prepared by the following stan-
dard procedure. A stirred solution of L-methionine (2.08 g)
in aqueous sodium hydroxide (40 mL, 2 M) at 0°C was
treated with methyl chloroformate (10.4 mL) in 1 mL incre-
ments over 1 h. The pH was maintained above 10.5 by addi-
tion of 2 M sodium hydroxide as required. After the final
aliquot of methyl chloroformate (0.4 mL) was added, the re-
action mixture was allowed to reach room temperature and
was then washed with an equal volume of ether. The aque-
ous layer was then cooled to 0°C, acidified to pH 2–3 with
10% HCl, and extracted with ethyl acetate (3 × 100 mL).
The extract was dried (MgSO4) and evaporated to yield N-
MOC-L-methionine (2.34 g, 84%) as an oil. NMR data are
listed below. Unless stated otherwise all products gave m/z
MH+ ions using FAB-NBA spectra.
Experimental
Materials and methods
Melting points were determined on a Kofler hot stage and
1
are uncorrected. The H NMR spectra were recorded on a
N-MOC-L- and D-methionines
Bruker Advance series 300 spectrometer in CDCl3 using re-
sidual CHCl3 as the internal standard or CD3OD using
CH3OH as the internal standard unless otherwise stated;
1H NMR (CDCl3) : 1.95–2.26 (m, 2H), 2.10 (s, 3H,
SCH3), 2.59 (t, 2H), 3.70 (s, 3H, OCH3), 4.50 (q, 1H), 5.39
© 2002 NRC Canada

636
Can. J. Chem. Vol. 80, 2002
(d, 1H), 6.47 (d, 1H). 13C NMR (CDCl3) : 15.6 (SCH3),
30.2 (SCH2), 31.9 (CH2), 53.0 (OCH3), 53.3 (CH), 157.5
(CO), 176.2 (CO). EI-MS m/z (%): 207 (26), 175 (40), 133
(47), 115 (58), 75 (66), 61 (100).
N-MOC-PRO-MET
1H NMR (CD3OD) : 1.8–2.3 (m, 8H), 2.09 (s, 3H,
SCH3), 2.60 (m, 2H), 3.64 (s, 3H, OCH3), 4.28 (m, 1H),
4.58 (m, 1H). 13C NMR (CD3OD) : 14.3, 24.4, 30.1, 30.4,
31.1, 51.7, 51.8, 60.2, 60.5, 156.3, 172.6, 174.3.
N-MOC-L- and D- ethionines
1H NMR (CDCl3) : 1.21 (t, 3H), 1.81–2.17 (m, 4H,
2CH2) 3.71 (s, 3H, OCH3), 4.34 (q, 1H), 4.84 (s, 1H), 7.35
(d, 1H). 13C NMR (CD3OD) : 14.1 (CH3), 25.4 (SCH2),
27.3 (SCH2), 31.6 (CH2), 52.6 (CH), 53.3 (OCH3), 163.5
(CO), 173.4 (CO).
N-MOC-PHE-MET
1H NMR (CD3OD) : 1.82–2.12 (m, 2H), 2.04 (s, 3H,
SCH3), 2.52 (m, 2H), 2.88 and 3.14 (m, total 2H), 3.58 (s,
3H, OCH3), 4.35 (m, 1H), 4.60 (m, 1H), 7.07 (d, 1H, NH),
7.24 (m, 5H), 8.40 (d, 1H, NH). 13C NMR (CD3OD) : 14.2,
30.0, 31.0, 38.0, 51.6, 51.7, 56.6, 126.7, 128.4 (2), 129.3 (2),
137.5, 157.3, 172.5, 173.4.
N-MOC-MET-GLY
1H NMR (CD3OD) : 1.8–2.1 (m, 2H, CH2), 2.08 (s, 3H,
SCH3), 2.55 (m, 2H), 3.34 (s, 3H, OCH3), 3.67 (br s, 2H),
4.28 (m, 1H), 5.6 (br s, 1H), 6.8 (br s, 1H). 13C NMR
(CD3OD) : 14.1 (SCH3), 30.0, 31.8, 40.9, 54.4, 56.4, 158.1,
171.9, 173.8.
Esterification
The esters of N-protected amino acids and dipeptides were
all obtained by the following standard procedure using the
appropriate alcohol. N-MOC-L-methionine (2.34 g) was dis-
solved in methanol (50 mL) and a catalytic amount of hy-
drochloric acid added to adjust the solution to pH 3.0. The
solution was heated under reflux for 12 h, then evaporated.
Water (50 mL) was added and the product extracted with
ethyl acetate. The solution was washed with saturated
NaHCO3, dried, and evaporated to give N-MOC-L-
methionine methyl ester as an oil (2.03 g, 81%). NMR data
are listed below. Unless listed otherwise, all products gave
m/z MH+ ions using FAB-NBA spectra.
N-MOC-MET-ALA
1H NMR (D2O) : 1.22 (d, 3H), 1.88 (m, 2H, CHCH3),
2.0 (s, 3H, SCH3), 2.51 (m, 2H), 3.57 (s, 3H, OCH3), 4.05
(q, 1H, CHCH3), 4.16 (m, 1H). 13C NMR (D2O) : 14.5,
17.8, 29.7, 31.0, 51.3, 53.0, 54.3, 159.3, 172.6, 173.2.
N-MOC-MET-LEU
1H NMR (CD3OD) : 0.75 (d, 3H, CHCH3), 0.81 (d, 3H,
CHCH3), 1.55 (m, 2H), 1.95–2.2 (m, 2H), 2.08 (s, 3H,
SCH3), 2.85 (m, 1H), 3.20 (m, 2H), 3.56 (s, 3H, OCH3),
4.17 (m, 1H), 4.34 (t, 1H). 13C NMR (D2O) : 13.8, 20.9
(2), 22.6, 24.5, 30.5, 39.9, 52.6, 52.9, 158.5, 173.8, 175.1.
N-Acetyl-L-methionine methyl ester
1H NMR (CD3OD) : 1.9–2.2 (m, 2H), 1.94 (s, 3H), 2.08
(s, 3H, SCH3), 2.42 (m, 2H), 3.60 (s, 3H, OCH3), 4.58 (m,
1H), 6.72 (d, 1H, NH). 13C NMR (CD3OD) : 14.3, 21.4,
30.1, 31.0, 51.8, 51.9, 172.4, 173.0.
N-MOC-MET-PRO
1H NMR (CD3OD) : 1.88–2.15 (m, 6H), 2.09 (s, 3H,
SCH3), 2.25 (m, 2H), 2.60 (t, 2H), 3.62 (s, 3H, OCH3), 4.49
(m, 1H), 4.62 (m, 1H). 13C NMR (CD3OD) : 14.3, 24.9,
29.0, 29.8, 31.1, 51.7, 59.4, 78.5, 156.8, 172.1, 173.0.
N-Chloroacetyl-L-methionine methyl ester
1H NMR (CDCl3) : 1.98–2.25 (m, 2H), 2.1 (s, 3H,
SCH3), 2.50 (t, 2H), 3.78 (s, 3H, OCH3), 4.08 (2H, s), 4.70
(m, 1H). 13C NMR (CDCl3) : 15.9, 30.2, 31.7, 42.8, 52.2,
53.1, 166.3, 172.1.
N-MOC-MET-PHE
1H NMR (CDCl3) : 1.8–2.0 (m, 2H), 2.08 (s, 3H, SCH3),
2.50 (m, 2H), 3.03 (m, 2H), 3.64 (s, 3H, OCH3), 4.22 (m,
1H), 4.72 (m, 1H), 7.23 (m, 5H). 13C NMR (CD3OD)
:
N-Formyl-L-methionine methyl ester
1H NMR (CDCl3) : 1.9–2.2 (m, 2H), 2.05 (s, 3H, SCH3),
2.52 (t, 2H), 3.73 (s, 3H, OCH3), 4.70 (m, 1H), 4.80 (s, 1H,
NH), 8.10 (s, 1H). 13C NMR (CDCl3) : 15.9, 30.2, 32.0,
50.6, 53.1, 161.1, 172.4.
14.2, 29.9, 31.9, 37.2, 51.7, 54.1, 54.3, 126.9, 128.5, 129.3,
137.0, 155.9, 172.2, 173.2.
N-MOC-GLY-MET
1H NMR (CD3OD) : 1.9–2.2 (m, 2H), 2.07 (s, 3H,
SCH3), 2.5–2.7 (m, 4H), 3.68 (s, 3H, OCH3), 4.57 (q, 1H).
13C NMR (CD3OD) : 14.2, 30.1, 31.1, 43.8, 51.5, 51.8,
157.7, 171.5, 171.8.
N-MOC-L- and D-methionine methyl esters
1H NMR (CDCl3) : 1.92–2.24 (m, 2H), 2.10 (s, 3H,
SCH3), 2.54 (t, 2H), 3.70 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 4.51 (q, 1H), 5.35 (d, 1H, NH). 13C NMR (CDCl3)
: 15.0, 29.6, 31.4, 52.0, 52.1, 52.8, 156.4, 172.4.
N-MOC-ALA-MET
1H NMR (CD3OD) : 1.32 (d, 3H), 1.9–2.2 (m, 2H), 2.09
(s, 3H, SCH3), 2.50 (m, 2H), 3.66 (s, 3H, OCH3), 4.12 (m,
1H), 4.58 (m, 1H). 13C NMR (CD3OD) : 14.2, 17.3, 30.1,
31.3, 50.9, 51.1, 51.6, 157.0, 173.8, 174.7.
N-MOC-L- and D-methionine ethyl esters
1H NMR (CDCl3) : 1.26 (t, 3H), 1.90–2.2 (m, 2H), 2.11
(s, 3H, SCH3), 2.52 (m, 2H), 3.67 (s, 3H, OCH3), 4.20 (2H,
q), 4.45 (m, 1H), 5.36 (m, 1H, NH). 13C NMR (CDCl3) :
14.5, 15.8, 30.2, 32.4, 52.7, 53.6, 62.0, 156.9, 172.4.
N-MOC-LEU-MET
1H NMR (CD3OD) : 0.96 (d, 3H), 0.98 (d, 3H), 1.58 (m,
2H), 1.70 (m, 1H), 1.9–2.2 (m, 2H), 2.05 (s, 3H, SCH3),
2.55 (m, 2H), 3.66 (s, 3H, OCH3), 4.16 (m, 1H), 4.57 (m,
1H). 13C NMR (CD3OD) : 14.2, 20.9, 22.3, 24.7, 30.1,
31.2, 41.0, 51.5, 51.6, 53.8, 156.8, 173.2, 174.6.
N-MOC-L- and D-methionine n-propyl esters
1H NMR (CDCl3) : 0.92 (t, 3H), 1.67 (sextet, 2H),
1.90–2.15 (m, 2H), 2.08 (s, 3H, SCH3), 2.51 (t, 2H), 3.68 (s,
3H, OCH3), 4.08 (q, 2H), 4.42 (m, 1H), 5.39 (m, 1H, NH).
© 2002 NRC Canada

Holland et al.
637
13C NMR (CDCl3) : 10.7, 15.8, 22.3, 30.3, 32.5, 52.7, 53.6,
67.6, 156.9, 172.5.
5H). 13C NMR (CD3OD) : 14.2, 29.9, 31.9, 37.2, 51.7 (2),
54.1, 54.3, 126.9, 128.5, 129.3, 137.0, 157.9, 172.2, 173.2.
N-MOC-GLY-MET methyl ester
N-MOC-L- and D-methionine n-butyl esters
1H NMR (CD3OD) : 1.94–2.2 (m, 2H), 2.07 (s, 3H,
SCH3), 2.56 (m, 2H), 3.67 (s, 3H, OCH3), 3.72 (s, 3H,
OCH3), 4.61 (m, 1H). 13C NMR (CD3OD) : 14.2, 30.0,
30.9, 43.7, 51.6, 51.8, 158.7, 171.3, 172.7.
1H NMR (CDCl3) : 0.92 (t, 3H), 1.33 (sextet, 2H), 1.60
(sextet, 2H), 1.87–2.15 (m, 2H), 2.07 (s, 3H, SCH3), 2.50 (t,
2H), 3.66 (s, 3H, OCH3), 4.10 (q, 2H), 4.41 (m, 1H), 5.40
(m, 1H, NH). 13C NMR (CDCl3) : 14.0, 15.8, 19.4, 30.3,
30.9, 32.5, 52.7, 53.6, 65.8, 156.9, 172.5.
N-MOC-ALA-MET methyl ester
1H NMR (CD3OD) : 1.33 (d, 3H), 1.9–2.18 (m, 2H),
2.07 (s, 3H, SCH3), 3.64 (s, 3H, OCH3), 3.73 (s, 3H, OCH3),
4.16 (m, 1H), 4.59 (m, 1H). 13C NMR (CD3OD) : 14.2,
17.3, 30.1, 31.3, 50.9, 51.1, 51.6 (2), 157.9, 173.8, 174.7.
N-MOC-L- and D-methionine n-pentyl esters
1H NMR (CDCl3) : 0.87 (t, 3H), 1.30 (m, 6H), 1.62 (m,
2H), 1.85–2.18 (m, 2H), 2.07 (s, 3H, SCH3), 2.49 (m, 2H),
3.64 (s, 3H, OCH3), 4.08 (q, 2H), 4.40 (m, 1H), 5.38 (m,
1H, NH). 13C NMR (CDCl3) : 14.3, 15.8, 22.6, 28.3, 28.6,
30.3, 32.5, 52.7, 53.6, 66.1, 156.9, 172.5.
N-MOC-LEU-MET methyl ester
1H NMR (CD3OD) : 0.92 (d, 3H), 0.94 (d, 3H), 1.54 (m,
2H), 1.70 (m, 1H), 1.9–2.18 (m, 2H), 2.05 (s, 3H, SCH3),
2.52 (m, 2H), 3.60 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 4.12
(m, 1H), 4.58 (m, 1H), 7.0 (d, 1H, NH), 8.35 (d, 1H, NH).
13C NMR (CD3OD) : 14.2, 21.0, 22.3, 24.8, 30.0, 30.9,
41.0, 51.6, 51.7, 53.7, 158.1, 172.6, 174.7.
N-MOC-L- and D- ethionine methyl esters
1H NMR (CD3OD) : 1.21 (t, 3H), 1.80–2.15 (m, 2H),
2.52 (m, 4H), 3.62 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 4.33
(m, 1H), 7.35 (m, 1H, NH). 13C NMR (CD3OD) : 14.1,
25.1, 27.5, 31.6, 51.7, 51.8, 53.3, 158.3, 173.4.
N-MOC-PRO-MET methyl ester
N-MOC-L-ethionine ethyl ester
1H NMR (CD3OD) : 1.82–2.3 (m, 8H), 2.1 (s, 3H,
SCH3), 2.60 (m, 2H), 3.64 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 4.28 (m, 1H), 4.58 (m, 1H). 13C NMR (CD3OD) :
14.3, 24.4, 30.1, 30.4, 31.1, 51.7, 51.8, 52.2, 60.2, 60.5,
156.0, 172.6, 174.3.
1H NMR (CD3OD) : 1.22 (t, 3H), 1.30 (t, 3H), 1.90–2.16
(m, 2H), 2.55 (m, 4H), 3.68 (s, 3H, OCH3), 4.21 (q, 2H),
4.35 (m, 1H). 13C NMR (CD3OD) : 13.5, 14.1, 25.5, 27.5,
31.6, 51.8, 71.6, 158.3, 173.0.
N-MOC-MET-GLY methyl ester
1H NMR (CDCl3) : 1.86–2.18 (m, 2H), 2.07 (s, 3H,
SCH3), 2.56 (m, 2H), 3.64 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 4.02 (m, 2H), 4.42 (m, 1H), 5.76 (m, 1H, NH), 6.98
(m, 1H, NH). 13C NMR (CD3OD) : 15.1, 31.0, 32.8, 41.8,
52.6, 52.8, 55.4, 159.1, 171.6, 175.1. EI-MS m/z (%): 278
(8), 246 (11), 204 (60), 172 (22), 90 (64), 61 (100),
N-MOC-PHE-MET methyl ester
1H NMR (CD3OD) : 1.82–2.1 (m, 2H), 2.06 (s, 3H,
SCH3), 2.52 (m, 2H), 2.87 (m, 1H), 3.14 (m, 1H), 3.58 (s,
3H, OCH3), 3.70 (s, 3H, OCH3), 4.35 (m, 1H), 4.60 (m, 1H),
7.25 (m, 5H), 8.39 (d, 1H, NH). 13C NMR (CD3OD) : 14.2,
30.0, 31.0, 38.0, 51.6, 51.7, 51.8, 56.6, 126.7, 128.4, 129.3,
137.4, 157.9, 172.5, 173.4.
N-MOC-MET-ALA methyl ester
1H NMR (D2O) : 1.38 (d, 3H), 1.82–2.04 (m, 2H), 2.01
(s, 3H, SCH3), 2.50 (m, 2H), 3.57 (s, 3H, OCH3), 3.68 (s,
3H, OCH3), 4.12 (q, 1H), 4.33 (m, 1H). 13C NMR (D2O) :
14.5, 16.3, 29.5, 31.0, 49.1, 53.0, 53.3, 54.2, 158.9, 174.6,
175.2.
Preparation of standard protected sulfoxides
Standard diastereomeric sulfoxide mixtures of esters of N-
protected amino acids and dipeptides were prepared using
the following representative procedure: N-MOC-L-
methionine methyl ester (2.03 g) was dissolved in methanol
(40 mL) in an Erlenmeyer flask (100 mL). In a separate
flask, hydrogen peroxide (1.13 g of 30% aqueous solution,
1.1 molar equivalents) was dissolved in methanol (15 mL).
Both solutions were cooled to –20°C, then mixed and al-
lowed to stand at –20°C overnight. The solvent was then re-
moved by vacuum to give a quantitative yield of a 1:1
diastereomeric mixture of N-MOC-L-methionine methyl es-
ter sulfoxides (oil).
N-MOC-MET-LEU methyl ester
1H NMR (CD3OD) : 0.74 (d, 3H, CHCH3), 0.81 (d, 3H,
CHCH3), 1.55 (m, 2H), 1.95–2.2 (m, 2H), 2.10 (s, 3H,
SCH3), 2.85 (m, 1H), 3.24 (m, 2H), 3.55 (s, 3H, OCH3),
3.60 (s, 3H, OCH3), 4.17 (m, 1H), 4.35 (t, 1H). 13C NMR
(D2O) : 13.6, 20.9 (2), 22.6, 24.5, 30.5, 39.9, 52.6, 52.9,
53.3, 158.8, 173.8, 175.1.
N-MOC-MET-PRO methyl ester
N-Acetyl-L-methionine methyl ester, sulfoxide
1H NMR (CD3OD) : 1.88–2.15 (m, 6H), 2.10 (s, 3H,
SCH3), 2.25 (m, 2H), 2.60 (t, 2H), 3.62 (s, 3H, OCH3), 3.80
(s, 3H, OCH3), 4.49 (m, 1H), 4.62 (m, 1H). 13C NMR
(CD3OD) : 14.3, 24.9, 29.0, 29.8, 30.1, 31.1, 51.7 (2), 59.4,
78.5, 158.1, 172.1, 173.0.
1H NMR (CD3OD) : 1.94 (s, 3H, COCH3), 2.2–2.4 (m,
2H), 2.52 (s, 3H, SOCH3), 2.8–3.0 (m, 2H), 3.60 (s, 3H,
OCH3), 4.62 (m, 1H). 13C NMR (CD3OD) : 21.4, 24.8,
37.2/37.3, 49.9, 51.6, 52.0, 172.0, 173.0.
N-Chloroacetyl-L-methionine methyl ester, sulfoxide
1H NMR (CD3OD) : 2.15 (m, 1H), 2.35 (m, 1H), 2.76 (s,
3H, SOCH3), 2.88 (m, 2H), 3.78 (s, 3H, OCH3), 4.10 (s,
2H), 4.60 (m, 1H). 13C NMR (CD3OD) : 24.7, 37.1/37.2,
41.9, 49.9, 52.0, 52.6, 166.8, 171.4.
N-MOC-MET-PHE methyl ester
1H NMR (CD3OD) : 1.7–2.1 (m, 2H), 2.02 (s, 3H,
SCH3), 2.48 (m, 2H), 3.05 (m, 2H), 3.64 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 4.22 (m, 1H), 4.72 (m, 1H), 7.25 (m,
© 2002 NRC Canada

638
Can. J. Chem. Vol. 80, 2002
N-Formyl-L-methionine methyl ester, sulfoxide
NMR (CD3OD) : 16.0, 24.71/24.79, 36.69/36.78, 49.65/
49.74, 52.90, 53.07, 53.64/53.86, 158.2, 172.3, 173.5.
1H NMR (CD3OD) : 2.0–2.3 (m, 2H), 2.62 (s, 3H,
SOCH3), 2.80 (m, 2H), 3.80 (s, 3H, OCH3), 4.80 (m, 1H),
8.22 (s, 1H). 13C NMR (CD3OD) : 25.8, 38.1/38.2,
50.71/50.75, 51.0, 53.1, 163.7, 172.4.
N-MOC-MET-LEU methyl ester, sulfoxides
1H NMR (CD3OD) : 0.75 (d, 3H), 0.81 (d, 3H),
1.50–1.60 (m, 3H), 2.58 (s, 3H, SOCH3), 2.75–2.90 (m, 2H),
3.53 (s, 3H, OCH3), 3.62 (s, 3H, OCH3), 4.16 (d, 1H), 4.34
(q, 1H). 13C NMR (CD3OD) : 20.6, 22.3 (2), 24.52/24.68,
37.06/37.13, 40.0, 50.6, 51.3, 51.8, 53.4, 157.9, 172.4,
173.5.
N-MOC-L- and D-methionine methyl esters, sulfoxides
1H NMR (CD3OD) : 2.2–2.6 (m, 2H), 2.66 (s, 3H,
SOCH3), 2.90 (m, 2H), 3.62 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 4.30 (m, 1H). 13C NMR (CD3OD) : 24.82/24.94,
37.1/37.25, 49.96/50.06, 52.0, 52.1, 53.2, 158.2, 172.4.
N-MOC-MET-PRO methyl ester, sulfoxides
N-MOC-L- and D-methionine ethyl esters, sulfoxides
1H NMR (CD3OD) : 1.28 (t, 3H), 2.2–2.58 (m, 2H), 2.60
(s, 3H, SOCH3), 2.94 (m, 2H), 3.68 (s, 3H, OCH3), 4.18 (q,
2H), 4.30 (m, 1H). 13C NMR (CD3OD) : 13.4, 24.72/24.95,
37.12/37.27, 48.8, 50.1, 51.73/51.83, 61.7, 158.3, 172.0.
1H NMR (CD3OD) : 1.88–2.15 (m, 4H), 2.40–2.60 (m,
2H), 2.60 (t, 2H), 2.70 (s, 3H, SOCH3), 2.90 (m, 2H), 3.62
(s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.50 (m, 1H), 4.62 (m,
1H). 13C NMR (CD3OD) : 24.8, 24.9, 29.0, 31.1,
37.23/37.34, 51.7 (2), 51.8, 59.4, 78.4, 158.1, 171.0, 172.9.
N-MOC-L- and D-methionine n-propyl esters, sulfoxides
1H NMR (CD3OD) : 0.94 (t, 3H), 1.67 (m, 2H), 2.2–2.5
(m, 2H), 2.68 (s, 3H, SOCH3), 2.80 (m, 2H), 3.62 (s, 3H,
OCH3), 4.08 (q, 2H), 4.44 (m, 1H), 7.32 (m, 1H, NH). 13C
NMR (CD3OD) : 9.8, 22.3, 24.70/24.94, 37.11/37.27,
51.71/51.80, 52.7, 53.6, 67.1, 158.3, 172.0.
N-MOC-MET-PHE methyl ester, sulfoxides
1H NMR (CD3OD) : 2.2–2.6 (m, 2H), 2.62 (s, 3H,
SOCH3), 2.88 (m, 2H), 3.06 (m, 2H), 3.65 (s, 3H, OCH3),
3.72 (s, 3H, OCH3), 4.22 (m, 1H), 4.72 (m, 1H), 7.25 (m,
5H). 13C NMR (CD3OD) : 25.56/25.61, 37.2, 37.31/37.39,
49.87/49.92, 51.7 (2), 54.1, 54.3, 126.9, 128.5, 129.3, 136.8,
157.9, 172.2, 173.3.
N-MOC-L- and D-methionine n-butyl esters, sulfoxides
1H NMR (CD3OD) : 0.95 (t, 3H), 1.40 (sextet, 2H), 1.60
(sextet, 2H), 2.2–2.6 (m, 2H), 2.68 (s, 3H, SOCH3), 2.90 (t,
2H), 3.70 (s, 3H, OCH3), 4.13 (q, 2H), 4.32 (m, 1H). 13C
N-MOC-GLY-MET methyl ester, sulfoxides
1H NMR (CD3OD) : 2.16 (m, 1H), 2.36 (m, 1H), 2.68 (s,
3H, SOCH3), 2.77–2.97 (m, 2H), 3.68 (s, 3H, OCH3), 3.78
NMR (CD3OD)
:
13.0, 19.4, 24.68/24.92, 32.5,
(s, 3H, OCH3), 3.80 (2H, s), 4.22 (m, 1H), 4.60 (m, 1H). 13
C
37.12/37.27, 50.2, 51.7, 53.4, 65.4, 158.3, 172.0.
NMR (CD3OD) : 24.8, 37.16/37.23, 43.8, 49.9, 51.3,
51.7/51.8, 52.0, 158.7, 171.7, 171.8.
N-MOC-L- and D-methionine n-pentyl esters, sulfoxides
1H NMR (CD3OD) : 0.87 (t, 3H), 1.30 (m, 6H), 1.62 (m,
2H), 2.22–2.5 (m, 2H), 2.63 (s, 3H, SOCH3), 2.65 (m, 2H),
3.62 (s, 3H, OCH3), 4.15 (q, 2H), 4.33 (m, 1H). 13C NMR
(CD3OD) : 14.2, 22.3, 24.69/24.93, 28.4, 31.0, 37.13/37.29,
50.10/50.16, 51.7, 53.4, 65.7, 157.6, 172.0.
N-MOC-ALA-MET methyl ester, sulfoxides
1H NMR (CD3OD) : 1.35 (d, 3H), 2.10 (m, 1H), 2.36 (m,
1H), 2.68 (s, 3H, SOCH3), 2.87 (m, 2H), 3.65, (s, 3H,
OCH3), 3.74 (s, 3H, OCH3), 4.10 (m, 1H), 4.60 (m, 1H). 13
C
NMR (CD3OD) : 17.1, 24.67/24.90, 37.18/37.27, 49.9,
50.8, 51.57/51.69, 52.0, 157.9, 171.6, 174.9.
N-MOC-L- and D- ethionine methyl esters, sulfoxides
1H NMR (CD3OD) : 1.33 (t, 3H), 2.11 (m, 1H), 2.33 (m,
1H), 2.72–2.97 (m, 4H), 3.64 (s, 3H, OCH3), 3.77 (s, 3H,
N-MOC-LEU-MET methyl ester, sulfoxides
OCH3), 4.34 (m, 1H). 13C NMR (CD3OD)
: 6.2,
1H NMR (CD3OD) : 0.92 (d, 3H), 0.94 (d, 3H), 1.54 (m,
2H), 1.70 (m, 1H), 2.2–2.45 (m, 2H), 2.66 (s, 3H, SOCH3),
2.82 (m, 2H), 3.63 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 4.15
(m, 1H), 4.58 (m, 1H). 13C NMR (CD3OD) : 21.6, 22.0,
25.5, 25.7, 38.14/38.20, 42.0, 50.9, 51.6, 51.7, 54.6, 159.1,
173.6, 175.6.
24.89/25.12, 45.3, 51.93/52.11, 52.3, 53.29, 53.62, 158.2,
172.5.
N-MOC-L-ethionine ethyl ester, sulfoxides
1H NMR (CD3OD) : 1.22 (t, 3H), 1.30 (t, 3H), 2.15 (m,
1H), 2.33 (m, 1H), 2.7–2.95 (m, 4H), 3.68 (s, 3H, OCH3),
4.20 (q, 2H), 4.36 (m, 1H). 13C NMR (CD3OD) : 6.1, 13.5,
24.88/25.11, 45.26/45.35, 51.71/51.81, 53.4, 53.5, 71.6,
158.3, 172.0.
N-MOC-PRO-MET methyl ester, sulfoxides
1H NMR (CD3OD) : 1.82–2.3 (m, 6H), 2.68 (s, 3H,
SOCH3), 2.80 (m, 2H), 3.62 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 4.30 (m, 1H), 4.58 (m, 1H). 13C NMR (CD3OD) :
23.5, 24.37/24.46, 30.4, 37.19/37.23, 49.97, 51.4, 51.8, 52.2,
60.2, 60.5, 156.3, 172.6, 174.4.
N-MOC-MET-GLY methyl ester, sulfoxides
1H NMR (CD3OD) : 2.0–2.32 (m, 2H), 2.66 (s, 3H,
SOCH3), 2.92 (t, 2H), 3.67 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 3.95 (q, 2H), 4.30 (m, 1H). 13C NMR (CD3OD) :
25.5, 37.08/37.14, 40.8, 49.63/49.77, 51.6, 51.8, 53.9, 157.9,
171.8, 173.0.
N-MOC-PHE-MET methyl ester, sulfoxides
1H NMR (CD3OD) : 2.25–2.60 (m, 2H), 2.66 (s, 3H,
SOCH3), 2.88 (m, 2H), 2.87 (m, 1H), 3.14 (m, 1H), 3.63 (s,
3H, OCH3), 3.75 (s, 3H, OCH3), 4.45 (m, 1H), 4.60 (m, 1H),
N-MOC-MET-ALA methyl ester, sulfoxides
1H NMR (CD3OD) : 1.30 (d, 3H), 1.98 (m, 1H), 2.10 (m,
1H), 2.58 (s, 3H, SOCH3), 2.75–2.90 (m, 2H), 3.55 (s, 3H,
OCH3), 3.60 (s, 3H, OCH3), 4.16 (d, 1H), 4.30 (q, 1H). 13C
7.25 (m, 5H). 13C NMR (CD3OD)
: 24.62/24.76,
37.51/37.60, 38.0, 50.08/50.16, 51.6 (2), 51.8, 56.8, 126.6,
128.4, 129.5, 137.6, 158.3, 172.3, 173.6.
© 2002 NRC Canada

Holland et al.
639
tals of L-methionine sulfoxide (62 mg, 76%), identified by
13C NMR and other analytical comparisons with authentic
samples (19) as (RSSC) material; mp230–235°C (lit. (25, 26)
mp 239°C). [ ]D –75 (c = 0.5, H2O) (lit. (25) [ ]D –77 (c =
0.5, H2O)).
Biotransformation procedures
Chloroperoxidase-catalyzed oxidations
The reactions were carried out using the following stan-
dard procedure: N-MOC-L-methionine methyl ester (33 mg,
0.15 mM) was dissolved in disodium citrate buffer
(16.6 mL, 0.1 M, pH 5.0) in a 50 mL flask. The solution was
stirred slowly at room temperature, and chloroperoxidase
(2000 units) was added. A solution of hydrogen peroxide
(56.6 L of 30% aqueous solution) in disodium citrate buffer
(1.6 mL, 0.1 M, pH 5.0) was then added via a syringe pump
over 70 min, with addition occurring directly below the sur-
face level of the reaction mixture. The reaction was stirred
for a further 5 min, quenched by addition of aqueous satu-
rated sodium sulfite (0.5 mL), and then continuously ex-
tracted with dichloromethane for 3 d. Evaporation of the
extract gave a quantitative recovery of amino acid material,
Acknowledgments
This work was funded by the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) and by a
grant from the American Chemical Society Petroleum Re-
search Fund B. We are grateful to Mr. T. Jones of Brock
University for help with the acquisition of spectral data.
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Ester hydrolysis of N-MOC-L-methionine methyl ester
sulfoxides
The diastereomeric mixture of chemically prepared N-
MOC-L-methionine methyl ester sulfoxide (100 mg) was
dissolved in phosphate buffer (0.1 M, pH 7.0, 20 mL), and
then the stirred solution was treated with the appropriate
Altus Chiroclec® enzyme (100 mg for each of
-
chymotrypsin, Aspergillus sp. protease, subtilisin Carlsberg,
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5–60
min,
monitored
by
TLC
(silica
gel,
EtOAc–MeOH–AcOH, 79:20:1), and on ca. 50% completion
the reaction was quenched by the addition of ethanol (5 mL)
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1
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above for N-MOC-L-methionine methyl ester sulfoxide and
N-MOC-L-methionine sulfoxide products. Configuration at
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Removal of N-MOC and ester-protecting groups
The following standard reaction procedure was used: N-
MOC-L-methionine methyl ester sulfoxide (120 mg), pre-
pared by CPO-catalyzed oxidation of N-MOC-L-methionine
methyl ester, was dissolved in a mixture of distilled water
(4 mL) and methanol (1 mL). Barium hydroxide (300 mg)
was added, and the mixture stirred and heated at reflux for
4 h. Water (10 mL) was added and the mixture was cooled
and adjusted to pH 5.75 with sulfuric acid (0.1 M), filtered
(celite), and evaporated to yield a semi-solid (100 mg),
which was then dissolved in distilled water (1 mL), filtered,
and the filtrate diluted with acetone (7 mL). The resulting
cloudy mixture was refrigerated overnight to produce crys-
26. F. Lucas and L. Levenbook. Biochem. J. 100, 473 (1966).
© 2002 NRC Canada