Biocatalytic and chemical routes to all the stereoisomers of methionine and ethionine sulfoxides

Article abstract of DOI:10.1016/S0957-4166(99)00271-2

Biotransformations of the N-phthaloyl derivatives of D- and L-methionine and of D- and L-ethionine by Beauveria bassiana ATCC 7159 or Beauveria caledonica ATCC 64970 produce the corresponding (S(s)) sulfoxides in good yield and diastereomeric excess. Pure

Full text of DOI:10.1016/S0957-4166(99)00271-2

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2833–2843
Biocatalytic and chemical routes to all the stereoisomers of
methionine and ethionine sulfoxides
Herbert L. Holland,∗ Peter R. Andreana and Frances M. Brown
Department of Chemistry, Brock University, St. Catharines, ON L2S 3A1, Canada
Received 27 April 1999; accepted 15 June 1999
Abstract
Biotransformations of the N-phthaloyl derivatives of D- and L-methionine and of D- and L-ethionine by
Beauveria bassiana ATCC 7159 or Beauveria caledonica ATCC 64970 produce the corresponding (SS) sulfoxides
in good yield and diastereomeric excess. Pure (SSSC) diastereomers can be obtained from L-series substrates by
crystallisation of the biotransformation extract, and the corresponding (SSRC) products obtained from D-series
substrates by chromatography of the biotransformation extract. Hydrogen peroxide-catalysed oxidation of the N-
phthaloyl derivatives of D- and L-methionine and of D- and L-ethionine gives diastereomeric mixtures from which
the (SSSC) and (RSRC) diastereomers can be obtained by crystallisation, and the (SSRC) and (RSSC) diastereomers
obtained by chromatography. N-Cbz- and N-t-Boc methionines are also converted to sulfoxides with predominant
(SS) configuration by both B. bassiana and B. caledonica, but the isolated yields and d.e. of products were generally
lower than those obtained from the N-phthaloyl substrates.
Removal of the N-phthaloyl group from diastereomerically pure methionine and ethionine sulfoxides gave
the corresponding amino acid sulfoxides in high yield; removal of N-Cbz and N-t-Boc groups from protected
methionine sulfoxides was also achieved without loss of configuration at sulfur. © 1999 Elsevier Science Ltd. All
rights reserved.
1. Introduction
Sulfoxides of the thia-amino acids methionine (1), ethionine (2), and (S)-methylcysteine (3) possess a
wide variety of biological properties. Oxidation of methionine residues in proteins has been the target of
considerable interest in recent years, as it often results in significant structural and functional changes in
the protein.1 This oxidation is reversible, and is performed by a methionine sulfoxide reductase enzyme
that functions as an in vivo antioxidant by transferring excess oxidising equivalents onto methionine
during times of cellular oxidative stress.2 The reversible oxidation of methionine is also implicated in
∗
Corresponding author. E-mail: holland@chemiris.labs.brocku.ca
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00271-2

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H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
other biological functions such as the modulation of potassium channel activity,3 and its role in the
oxidation of high density lipoproteins, leading to a reduced ability of the latter to transport cholesterol
to the liver for excretion, has recently been established.4,5 It is also implicated in the ageing process by
demonstration of the effect of methionine sulfoxidation on the activity of the human keratinocyte growth
factor6 and on the immuno-peptide thymosin β-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 responsible for the anti-bacterial action of cabbage10
and possesses the ability to lower serum cholesterol levels.11 It is also the substrate for the enzyme
cysteine sulfoxide lyase, whose activity in damaged vegetable tissue leads to the production of the volatile
sulfur compounds responsible for the characteristic ‘rotting vegetable’ smells.12
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 (SSSC) configuration, no data are
available on the configuration at sulfur in naturally occurring amino acid sulfoxides from other sources.
Prior to our preliminary report, no diastereoselective method for the oxidation of a sulfur-containing
amino acid to its sulfoxide appears in the literature.15 The potential relevance of the configuration
at sulfur to the biological activity of oxidised amino acids is clearly indicated by a report on the
differential toxicity of the diastereomers of an (S)-pentachlorobutadienylcysteine sulfoxide metabolite
of hexachlorobutadiene.16
The value of biocatalytic oxidation as a method for the stereoselective oxidation of sulfides to
sulfoxides is well established,17,18 and this method has been used for the oxidation of methionine by
Penicillium citreo-viride, but no stereochemical details were reported.19 In view of the ability of fungal
biocatalysts to oxidise a wide variety of prochiral sulfides to chiral sulfoxides, we have investigated such
catalysts for the diastereoselective oxidation of a range of N-protected thia-amino acids, and now report
their use for the diastereoselective oxidation of methionine and ethionine derivatives which, together with
complementary chemical oxidations, can lead to the preparation of all the stereoisomers of methionine
and ethionine sulfoxides.
2. Results
Screening experiments for the ability of fungi to oxidise N-phthaloyl-D,L-methionine were carried
out using established oxidative biocatalysts18,20 such as Aspergillus niger ATCC 9142, Aspergillus
ochraceous ATCC 18500, Curvularia lunata ATCC 12017, Mortierella isabellina ATCC 42613, Hel-
minthosporium species NRRL 4671, Rhizopus arrhizus ATCC 11145, and Beauveria bassiana ATCC
7159. Of these, only the latter gave sulfoxide product in >10% conversion, producing in unoptimised
biotransformation of N-phthaloyl-L-methionine an 85% yield of a product subsequently identified as
predominantly the (SSSC) sulfoxide (d.e. 60%), and from N-phthaloyl-D-methionine 88% of the (SSRC)
sulfoxide (d.e. 74%), as summarised in Table 1. This led to the investigation of other Beauveria species,
and B. caledonica ATCC 64970 was identified as a superior catalyst for the oxidation of N-phthaloyl-L-
methionine to the (SSSC) sulfoxide, giving a quantitative conversion to the (SSSC) sulfoxide (d.e. 90%),
while N-phthaloyl-D-methionine was converted to the (SSRC) sulfoxide in lower yield but with high d.e.
(92%) (Table 1).

H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
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Table 1
Sulfoxide products obtained from the biotransformation of N-protected methionine substrates
Table 2
Sulfoxide products obtained from the biotransformation of N-phthaloylethionine substrates
The configuration of the predominant diastereomer obtained from biocatalytic oxidations of N-
phthaloyl-L-methionine was determined following its crystallisation to homogeneity, comparison of
analytical data with those reported for (SSSC)-N-phthaloylmethionine sulfoxide,21 and conversion
to (SSSC)-methionine sulfoxide22 by hydrazinolysis. The product obtained from N-phthaloyl-D-
methionine was diastereomeric with that obtained from the L-isomer, and thus identified as (SSRC)-
N-phthaloylmethionine sulfoxide, subsequently converted to (SSRC)-methionine sulfoxide.22 Other
N-protected methionine substrates, also summarised in Table 1, gave consistently lower yields or
diastereomeric purities of non-crystalline products which were not readily separable into individual
diastereomers by chromatography, and were thus characterised following removal of the protecting
group by comparison with authentic standards of methionine sulfoxides. Mixtures of the latter give
baseline-resolved 13C NMR signals for the individual diastereomers at 75 MHz.
In view of the superior results obtained from N-phthaloylmethionines, only this protecting group
was used for investigation of the biotransformation of N-protected L- and D-ethionine substrates. The
results, summarised in Table 2, parallel those obtained from the corresponding methionines in terms of
stereoselectivity of oxidation, but in this instance no significant differences were observed between the
two Beauveria strains employed.
The product from biotransformations of N-phthaloyl-L-ethionine could be crystallised to homogeneity,
and was identified as the (SSSC) diastereomer by determination of its X-ray crystallographic structure
(Fig. 1). The product from N-phthaloyl-D-ethionine could not be purified by crystallisation, but chroma-

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H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
Figure 1. (SSSC)-N-Phthaloylethionine
tography gave a 46% yield of sulfoxide, d.e. 95%, which was diastereomeric with that obtained from the
L-isomer of substrate, and thus identified as (SSRC)-N-phthaloylethionine.
N-Phthaloyl-L- and D-methionine and N-phthaloyl-L- and D-ethionine were all oxidised by hydrogen
peroxide non-stereoselectively but in quantitative yields to the corresponding sulfoxides. From these
mixtures, the (SSSC) and (RSRC) diastereomers could be obtained in pure form by crystallisation, and the
(SSRC) and (RSSC) diastereomers obtained from the corresponding mother liquors by chromatography.
Removal of the N-phthaloyl group from N-phthaloylmethionine and -ethionine sulfoxides was achie-
ved by hydrazinolysis, avoiding the classical acidic work-up that leads to racemisation of the configu-
ration at sulfur, and yielded the corresponding amino acid sulfoxides in good yield. The configurations
of products in the methionine series were determined by comparison with literature data for standards
of known configuration, and configurations in the ethionine series were based on correlations with the
structure of Fig. 1.
3. Discussion
A flow chart for the preparation of methionine and ethionine sulfoxides is presented in Scheme 1,
below.
Scheme 1. Summary of routes for the preparation of methionine and ethionine sulfoxides

H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
2837
Specific biotransformation yields and percentage compositions given in Scheme 1 refer to conversions
of N-phthaloyl-L- and D-methionines by B. caledonica, but the stereochemical relationships outlined in
the Scheme are relevant to all the isomers of N-phthaloylmethionines and -ethionines. The stereostruc-
tures are shown below for methionine (R=CH3) and ethionine (R=C2H5) sulfoxides.
The routes outlined in Scheme 1 for the preparation of diastereomerically defined N-phthaloyl-
methionine and -ethionine sulfoxides, and thence for the corresponding amino acid sulfoxides, present a
simple method for the preparation of the latter, and are superior to existing resolution-based methods for
the preparation of the individual diastereomers of methionine sulfoxide.22 No methods for the preparation
of the corresponding ethionine sulfoxides have previously been reported.
The biocatalytic approach is currently being extended to the preparation of other naturally occurring
amino acid sulfoxides, and these results will be presented in due course.
4. Experimental
4.1. Materials and methods
Melting points were determined on a Kofler hot stage and are uncorrected. The 1H NMR spectra were
recorded on a Bruker Avance series 300 spectrometer in CDCl3 using residual CHCl3 as the internal
standard unless otherwise stated; chemical shifts are reported in ppm (δ) 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 solution as stated below. Mass spectra
were obtained in EI mode (unless otherwise stated) using a Kratos 1S spectrometer. IR spectra were
obtained using a Mattson Research Series FTIR spectrometer. Optical rotations were recorded at ambient
temperature in the stated solvent using a Rudolph Autopol 3 polarimeter. Diastereomeric excess (d.e.) was
determined by 13C NMR analysis of signals α and β to sulfur. TLC was performed on Merck silica gel
F
254 plates, 0.2 mm, and column chromatography used Merck silica gel 9385, 230–400 mesh. Beauveria
bassiana ATCC 7159 was maintained on Sabouraud dextrose agar slopes, grown at 26°C and stored at
4°C, and Beauveria caledonica ATCC 64970 on potato dextrose agar slopes, grown at 24°C and stored
at 4°C.
4.2. Preparation of substrates
N-Cbz- and N-t-Boc-methionines were commercial samples (Sigma). N-Phthaloyl-D- and L-
methionines and D- and L-ethionines were prepared from the amino acids in 95–98% yield by the
method of Bose.23
1
N-Phthaloyl-D-methionine: m.p. 123–124°C; H NMR δ 2.08 (3H, s), 2.42–2.62 (4H, m), 5.15 (1H,
m), and 7.75/7.89 (each 2H, m) ppm; 13C NMR δ (CDCl3) 15.7, 28.2, 31.2, 51.0, 124.1, 132.1, 134.7,
167.9, and 175.1 ppm; MS m/z (%) 279 (12), 205 (100), 187 (86), 132 (29); [α]D +47.46 (c 5.0, MeOH).
N-Phthaloyl-L-methionine: m.p. 122–124°C (lit.24 m.p. 124°C); spectral data as above; [α]D −47.50
(c 5.0, MeOH) (lit.25 [α]D −47.5 (c 5.2, MeOH)).

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H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
1
N-Phthaloyl-D-ethionine: m.p. 77–79°C; H NMR δ 1.22 (3H, t), 2.38–2.66 (6H, m), 5.15 (1H, m),
7.70/7.90 (each 2H, m), and 8.85 (1H, br.s) ppm; 13C NMR δ (CDCl3) 15.0, 26.1, 28.7, 28.8, 51.1, 124.0,
132.1, 134.7, 167.9, and 174.8 ppm; MS m/z (%) 293 (13), 205 (100), 187 (97), 132 (24); [α]D +45.7 (c
1.5, MeOH); calcd for C14H15NO4S C 57.32, H 5.15, N 4.77%, found C 57.55, H 5.14, N 4.77%.
N-Phthaloyl-L-ethionine: m.p. 78–80°C; spectral data as above; [α]D −47.2 (c 2.5, MeOH); calcd for
C14H15NO4S C 57.32, H 5.15, N 4.77%, found C 57.33, H 5.15, N 4.69%.
4.3. Biotransformation procedures
4.3.1. B. bassiana
A growth medium (3 L) composed of glucose (10 g) and corn steep liquor (20 g) per L of distilled
water, adjusted to pH 4.85 with 1 M NaOH was distributed in 15 1 L Erlenmeyer flasks which were
stoppered with foam plugs and sterilised by autoclaving at 121°C for 20 min. The flasks were allowed
to cool, then inoculated under sterile conditions with B. bassiana taken from a 3-day-old agar slope. The
flasks were allowed to stand overnight at 27°C, then placed on a rotary shaker (1 in. orbit) at 180 rpm,
27°C. After 3 days, a solution of the appropriate substrate (1.0 g) in 95% ethanol (30 mL) was added,
and growth allowed to continue for a further 3 days. The fungal mass was removed by filtration, and the
filtrate adjusted to pH 3 and continuously extracted with dichloromethane for 4 days. The extract was
treated with decolourising carbon in chloroform solution, and then evaporated to give a residue that was
treated as described below.
4.3.2. B. caledonica
This was grown in 1 L Erlenmeyer flasks as described above in a medium of potato dextrose broth at
24–26°C. The substrate was added 6 days after inoculation of the culture, and biotransformation allowed
to proceed for a further 3 days. Subsequent procedures were as described above. B. caledonica could
also be grown and used for biotransformation in the glucose/corn steep liquor medium described for
B. bassiana. No qualitative or quantitative differences in product produced using the two media were
observed.
4.4. Characterisation of biotransformation products
Spectral data are listed below under the appropriate substrate headings for products from biotransfor-
mations with B. bassiana. The yield and d.e. of products from B. caledonica are listed in Tables 1 and
2.
4.4.1. N-Phthaloyl-D-methionine
(SSRC)-N-Phthaloylmethionine sulfoxide. The crude biotransformation extract from B. bassiana (1.8
1
g from 2 g substrate) was analysed by H and 13C NMR and found to consist largely of N-phthaloyl-
D-methionine sulfoxide, d.e. 74%. The material resisted crystallisation, and was purified by chromato-
graphy using an ethyl acetate/methanol gradient elution to yield 65% of material with d.e. 95% (13C
NMR), m.p. 180–183°C; 1H NMR δ 2.45–2.8 (2H, m), 2.70 (3H, s), 2.9–3.05 (2H, m), 5.0 (1H, m), and
7.75/7.88 (each 2H, m) ppm; 13C NMR δ (CD3OD) 23.03, 36.98, 51.03, 52.75, 123.30, 132.38, 134.51,
168.64, and 174.41 ppm; MS m/z (%) 295 (2), 279 (11), 232 (18), 205 (100), 187 (95), 132 (33); [α]D
+63.0 (c 0.96, EtOH), determined following conversion to methionine sulfoxide (see below) to be the
(SSRC) diastereomer.

H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
2839
4.4.2. N-Phthaloyl-L-methionine
(SSSC)-N-Phthaloylmethionine sulfoxide. The crude biotransformation extract from B. bassiana (3.5
g from 4 g substrate) was analysed by 1H and 13C NMR and found to consist largely of N-phthaloyl-L-
methionine sulfoxide, d.e. 60%. The material was crystallised by dissolving in hot methanol (30 mL), the
volume of the solution reduced to ca. 15 mL by boiling, and the solution then allowed to cool to room
temperature to yield 2.56 g (64%) of material with d.e. ≥95% (13C NMR), m.p. 216–218°C (lit.21 m.p.
215–217°C); 1H NMR δ 2.45–2.8 (2H, m), 2.70 (3H, s), 2.9–3.05 (2H, m), 4.90 (1H, m), and 7.75/7.88
(each 2H, m) ppm; 13C NMR δ (CD3OD) 23.25, 37.24, 51.37, 53.0, 123.29, 132.37, 134.51, 168.63, and
174.41 ppm; MS m/z (%) 295 (2), 279 (14), 232 (18), 205 (100), 187 (93), 132 (34); [α]D +12.5 (c 0.65,
EtOH) (lit.21 [α]D +13 (c 0.35, EtOH)), confirmed by conversion to methionine sulfoxide (see below) as
the (SSSC) diastereomer.
4.4.3. N-Phthaloyl-D-ethionine
(SSRC)-N-Phthaloylethionine sulfoxide. The crude biotransformation extract from B. bassiana (1.52 g
from 1.5 g substrate) was analysed by 1H and 13C NMR and found to consist largely of N-phthaloyl-D-
ethionine sulfoxide, d.e. 76%. The material resisted crystallisation, and was purified by chromatography
using an ethyl acetate/methanol gradient elution to yield 46% of material with d.e. 95% (13C NMR), m.p.
163–165°C; 1H NMR δ (CD3OD) 1.30 (3H, t), 2.58–3.0 (6H, m), and 4.78 (1H, m), and 7.80/7.90 (each
2H, m) ppm; 13C NMR δ (CD3OD) 6.10, 22.77, 45.22, 47.90, 51.09, 123.47, 132.12, 134.73, 168.19,
and 170.54 ppm; MS (FAB-NBA) m/z (%) 310 (M+H, 80), 288 (72), 232 (52), 186 (94), 176 (100); [α]D
+41.4 (c 1.14, MeOH); calcd for C14H15NO5S C 54.36, H 4.89, N 4.53%, found C 54.12, H 5.16, N
4.46%.
4.4.4. N-Phthaloyl-L-ethionine
(SSSC)-N-Phthaloylethionine sulfoxide. The crude biotransformation extract from B. bassiana (2.02
1
g from 2 g substrate) was analysed by H and 13C NMR and found to consist largely of N-phthaloyl-
L-ethionine sulfoxide, d.e. 86%. The material was crystallised by dissolving in hot methanol (30 mL),
the volume of the solution reduced to ca. 15 mL by boiling, and the solution then allowed to cool to
1
room temperature to yield 1.1 g (50%) of material with d.e. ≥95% (13C NMR), m.p. 218–220°C; H
NMR δ 1.32 (3H, t), 2.5–3.05 (6H, m), 4.94 (1H, m), and 7.72/7.88 (each 2H, m) ppm; 13C NMR δ
5.98, 22.94, 45.35, 47.80, 51.52, 123.47, 132.12, 134.73, 168.19, and 170.54 ppm; MS (FAB-NBA) m/z
(%) 310 (M+H, 100), 232 (46), 186 (44); [α]D −21.1 (c 0.7, EtOH); calcd for C14H15NO5S C 54.36, H
4.89, N 4.53%, found C 54.34, H 5.06, N 4.54%, identified by crystallography as the (SSSC) diastereomer.
Material for X-ray crystallography was obtained by slow evaporation of a methanol/ethyl acetate solution
to give monoclinic crystals, space group P21, a=5.4708(15) Å, b=9.783(4) Å, c=13.341(4) Å, α=90°,
β=91.489(19)°, γ=90°, V=713.8(4) Å3. Details are deposited in the Cambridge Crystallographic Data
Centre and are available as supplementary information.
4.4.5. N-Cbz-D-methionine
(SSRC)-N-Cbz-D-methionine sulfoxide. The crude biotransformation product (1.04 g from 1 g subs-
trate), d.e. 32% (13C NMR), was subjected to chromatography using an ethyl acetate/methanol gradient
elution to yield 46% of material with d.e. 30% (13C NMR); oil; 1H NMR δ 2.22–2.38 (2H, m), 2.57/2.63
(total 3H, ratio 35:65, s), 2.81–2.88 (2H, m), 4.47 (1H, m), 5.09 (2H, s), 6.06/6.16 (total 1H, d, exchanges
D2O), 7.34 (5H, m), and 9.30 (1H, br.s, exchanges D2O) ppm; 13C NMR δ (CDCl3), 25.84/26.26 (35:65),
37.54/37.70 (35:65), 49.45, 53.04/53.29 (65:35), 67.53, 128.58, 128.63, 128.96, 136.54, 156.54, and

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H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
173.51/175.04 ppm; IR νmax 3200–3500, 2900–3100, 1700, 1600, and 1050 cm−1, determined following
conversion to methionine sulfoxide (see below) to be the (SSRC) diastereomer.
4.4.6. N-Cbz-L-methionine
(SSSC)-N-Cbz-L-methionine sulfoxide. The crude biotransformation product (0.544 g from 0.5 g
substrate), d.e. 40% (13C NMR), was subjected to chromatography using an ethyl acetate/methanol
1
gradient elution to yield 46% of material with d.e. 40% (13C NMR); oil; H NMR as in the preceding
paragraph except for δ 2.57/2.63 (total 3H, ratio 70:30); 13C NMR δ (CDCl3), 25.84/26.26 (70:30),
37.54/37.70 (70:30), 49.45, 53.04/53.29 (30:70), 67.53, 128.58, 128.63, 128.96, 136.54, 156.54, and
173.51/175.04 ppm; IR as above, determined following conversion to methionine sulfoxide (see below)
to be the (SSSC) diastereomer.
4.4.7. N-t-Boc-D-methionine
(SSRC)-N-t-Boc-D-methionine sulfoxide. The crude biotransformation product (0.72 g from 1 g
substrate), d.e. 60% (13C NMR), was subjected to chromatography using an ethyl acetate/methanol
1
gradient elution to yield 41% of material with d.e. 60% (13C NMR); oil; H NMR δ 1.43 (9H, s), 2.25
(2H, m), 2.68/2.71 (total 3H, ratio 22:78, s), 2.8 (2H, m), 4.16 (1H, m), and 6.32 (1H, br.s, exchanges
D2O) ppm; 13C NMR δ (CDCl3) 25.01/26.28 (21:79), 28.55, 37.23/38.0 (20:80), 49.22/49.82 (19:81),
53.92, 79.09, 155.93, and 177.57 ppm, determined by conversion to methionine sulfoxide (see below) to
be the (SSRC) diastereomer.
4.4.8. N-t-Boc-L-methionine
(SSSC)-N-t-Boc-L-methionine sulfoxide. The crude biotransformation product (0.6 g from 1 g subs-
trate), d.e. 58% (13C NMR) was subjected to chromatography using an ethyl acetate/methanol gradient
1
elution to yield 45% of material with d.e. 60% (13C NMR); oil; H NMR δ 1.44 (9H, s), 2.24–2.36
(2H, m), 2.66/2.71 (total 3H, ratio 80:20, s), 2.88–2.94 (2H, m), 4.41 (1H, m), and 5.66/5.73 (total 1H,
br.s, exchanges D2O); 13C NMR δ (CDCl3) 25.01/26.28 (78:22), 28.55, 37.23/38.0 (79:21), 49.22/49.82
(81:19), 54.06, 79.08, 155.93, and 177.48 ppm, determined by conversion to methionine sulfoxide (see
below) to be the (SSSC) diastereomer.
4.5. Hydrogen peroxide oxidations
4.5.1. N-Phthaloyl-D-methionine
Separate solutions of N-phthaloyl-D-methionine (2.79 g, 10 mmol) in methanol (50 mL) and hydrogen
peroxide (1.246 g of 30 wt%, 11 mmol) in methanol (50 mL) were cooled to −20°C and then mixed. The
mixture was allowed to stand at −20°C overnight, then evaporated to dryness to give 2.95 g (100%) of
crude product, d.e. ca. 0%. This was dissolved in 10 mL of boiling methanol, and the solution allowed
to cool to room temperature, yielding 1.3 g of product, d.e. 80–85%. One further recrystallisation from
methanol (10 mL) then afforded 0.85–0.9 g of diastereomerically pure product, m.p. 217–218°C, [α]D
−12.5 (c 0.585, EtOH), spectral data identical with those reported above for the (SSSC) diastereomer,
determined following conversion to methionine sulfoxide (see below) to be the (RSRC) diastereomer.
Evaporation of the mother liquors gave a sample enriched in the (SSRC) diastereomer (d.e. 76%),
whose purity remained unchanged on crystallisation from a wide variety of solvents, but which, on
chromatography as described above, gave the (SSRC) diastereomer, m.p. 179–181°C, spectral data as
described above, [α]D +62.5 (c 1.1, EtOH), d.e. 95%.

H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
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4.5.2. N-Phthaloyl-L-methionine
Oxidation of N-phthaloyl-L-methionine by the procedure in the preceding paragraph, followed by
purification of the product as described, gave 0.85–0.9 g of diastereomerically pure sulfoxide, m.p.
and spectral data identical with those reported above and previously reported,21 determined following
conversion to methionine sulfoxide (see below) to be the (SSSC) diastereomer. Evaporation of the mother
liquors gave a sample enriched in the (RSSC) diastereomer (d.e. 72%), whose purity remained unchanged
on crystallisation from a wide variety of solvents, but which, on chromatography as described above,
gave the (RSSC) diastereomer, m.p. 180–183°C, spectral data identical with those described above, [α]D
−61.8 (c 1.25, EtOH), d.e. 95%.
4.5.3. N-Phthaloyl-D-ethionine
Separate solutions of N-phthaloyl-D-ethionine (1.354 g) in methanol (25 mL) and hydrogen peroxide
(0.74 g of 30 wt%) in methanol (25 mL) were cooled to −20°C and then mixed. The mixture was allowed
to stand at −20°C overnight, then evaporated to dryness to give 1.45 g (100%) of crude product, d.e.
ca. 0%. This was dissolved in 8 mL of boiling methanol, and the solution allowed to cool to room
temperature, yielding 0.6 g of product, d.e. 80–85%. One further recrystallisation from methanol (3 mL)
then afforded 0.4 g of a diastereomerically pure product, m.p. 217–219°C, spectral data as reported above
for the (SSSC) isomer, [α]D +21.0 (c 0.71, EtOH), which was determined to be the (RSRC) isomer by
comparison with the authentic (SSSC) isomer. Evaporation of the combined mother liquors gave material
enriched in the (SSRC) diastereomer, d.e. 70%, whose purity remained unchanged on crystallisation
from a wide variety of solvents, but which, on chromatography as described above, gave the (SSRC)
diastereomer, m.p. 165–168°C, spectral data identical with those described above, [α]D +37.8 (c 1.33,
EtOH), d.e. 95%.
4.5.4. N-Phthaloyl-L-ethionine
Oxidation as described in the preceding paragraph gave a comparable yield of the (SSSC) isomer, m.p.
218–220°C, [α]D −21.4 (c 0.95, EtOH), identified by comparison with an authentic sample (above).
Evaporation of the combined mother liquors gave material enriched in the (RSSC) diastereomer, d.e.
74%, whose purity remained unchanged on crystallisation from a wide variety of solvents, but which,
on chromatography as described above, gave the (RSSC) diastereomer, m.p. 164–166°C, spectral data
identical with those described above, [α]D −39.8 (c 1.35, EtOH), d.e. 95%.
4.6. Removal of protecting groups
4.6.1. N-Phthaloylmethionine sulfoxides
A solution of (SSSC)-N-phthaloylmethionine sulfoxide (0.45 g) in 95% ethanol (10 mL) was treated
with hydrazine hydrate (0.10 g, 95%), and the resulting mixture stirred and heated to reflux on a water
bath for 2 h. The mixture was then evaporated to dryness, dissolved in distilled water (10 mL), and
filtered. The filtrate was evaporated to give 0.27 g of material, which was redissolved in distilled water (4
mL) and the resulting solution clarified by filtration through a 0.45 µ Millipore™ filter. Acetone (25 mL)
was added to the filtrate and the mixture was allowed to stand overnight at room temperature. The product
was then removed by filtration, washed with acetone and vacuum dried to give 0.18 g (72%) of (SSSC)-
methionine sulfoxide, m.p. >260°C (dec.) (lit.22,26 m.p. 250–255°C (dec.)); 1H NMR δ (D2O) 2.13–2.32
(2H, m), 2.59 (3H, s), 2.78–3.08 (2H, m), and 4.10 (1H, m) ppm; 13C NMR δ (D2O) 23.49, 37.01, 48.26,
51.97, and 171.09 ppm; [α]D +98.2 (c 0.5, H2O) (lit.22 +99.7 (c 0.5, H2O)). Also obtained from the
corresponding N-phthaloyl material by an identical procedure were (RSRC)-methionine sulfoxide, m.p.

2842
H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
>260°C (dec.) (lit.22,26 m.p. 250–255°C (dec.) for the (SSSC) enantiomer); spectral data identical with
those reported above; [α]D −99.3 (c 0.7, H2O), (RSSC)-methionine sulfoxide, m.p. 235°C (dec.) (lit.22,26
m.p. 239°C (dec.); 1H NMR δ (D2O) 2.13–2.32 (2H, m), 2.59 (3H, s), 2.78–3.08 (2H, m), and 4.10 (1H,
m) ppm; 13C NMR δ (D2O) 23.71, 37.01, 48.38, 52.09, and 171.09 ppm; [α]D −78 (c 0.5, H2O) (lit.22
−77 (H2O)), and (SSRC)-methionine sulfoxide, m.p. 235–240°C (dec.) (lit.22,26 m.p. 239°C (dec.) for the
(RSSC) enantiomer); spectral data identical with those reported above; [α]D +78 (c 0.5, H2O).
4.6.2. N-Phthaloylethionine sulfoxides
A solution of (SSSC)-N-phthaloylethionine sulfoxide (0.373 g) in 95% ethanol (10 mL) was treated
with hydrazine hydrate (0.075 g, 95%), and subsequent procedures carried out as described above for
the preparation of (SSSC)-methionine sulfoxide. After purification the product (0.152 g, 77%) had m.p.
1
247–250°C (dec.); H NMR δ (D2O) 1.14 (3H, t), 2.12 (2H, m), 2.65–2.9 (4H, m), and 3.70 (1H,
m) ppm; 13C NMR δ (D2O) 6.31, 24.26, 44.76, 45.90, 53.66, and 173.52 ppm; [α]D +50.0 (c 1.04,
H2O); calcd for C6H13NO3S C 40.21, H 7.31, N 7.81%, found C 40.28, H 7.68, N 7.91%. Also obtained
from the corresponding N-phthaloyl material by an identical procedure were (RSRC)-ethionine sulfoxide,
m.p. 250°C (dec.); spectral data identical with those reported above; [α]D −49.3 (c 0.5, H2O), (RSSC)-
ethionine sulfoxide, m.p. 219–221°C (dec.); 1H NMR δ (D2O) 1.18 (3H, s), 2.08–2.25 (2H, m), 2.68–3.0
(4H, m), and 3.70 (1H, m) ppm; 13C NMR δ (D2O) 6.34, 24.48, 44.64, 45.96, 53.97, and 173.54 ppm;
[α]D −13.2 (c 0.7, H2O); calcd for C6H13NO3S C 40.21, H 7.31, N 7.81%, found C 40.04, H 7.30,
N 7.82%, and (SSRC)-methionine sulfoxide, m.p. 219–221°C (dec.); spectral data identical with those
reported above; [α]D +12.7 (c 0.8, H2O).
4.6.3. N-Cbz-methionine sulfoxides
A solution of N-Cbz-L-methionine sulfoxide obtained by biotransformation of N-Cbz-L-methionine
using B. bassiana (53 mg, d.e. 40%) in dry chloroform (3 mL) under argon at room temperature
was treated with 65 µL of TMS iodide (1.2 equiv.), and the reaction warmed and monitored for the
disappearance of starting material by TLC (typically 3–5 h at 35–45°C). On completion, the reaction
was quenched by the addition of methanol (260 µL), stirred for a further 5 min, then evaporated to
dryness. The residue was dissolved in a mixture of ether (3 mL) and 1 M H2SO4 (3 mL), and the aqueous
layer washed with ether (3×3 mL). The aqueous layer was then adjusted to pH 5.75 with 1 M Ba(OH)2,
clarified by filtration (Celite and Millipore™ filtration) and evaporated to dryness to give a sample of
methionine sulfoxide (22.5 mg, 80%), determined by 13C NMR analysis (D2O) by comparison and in
admixture with authentic diastereomers to consist of a mixture of (SSSC) and (RSSC) diastereomers in
the ratio 70:30. Deprotection of N-Cbz-D-methionine sulfoxide (from biotransformation of N-Cbz-D-
methionine by B. bassiana) was performed in an analogous way.
4.6.4. N-t-Boc-methionine sulfoxides
A solution of N-t-Boc-L-methionine sulfoxide obtained by biotransformation using B. bassiana (116
mg, d.e. 58%) in water (5 mL) was treated with 2.6 mL (6 equiv.) of 1 M H2SO4. The resulting solution
was stirred overnight at room temperature, then adjusted to pH 5.75 with 1 M Ba(OH)2, clarified by
filtration (Celite and Millipore™ filtration) and evaporated to dryness to give a sample of methionine
sulfoxide (62 mg, 85%), determined by 13C NMR analysis (D2O) by comparison and in admixture
with authentic diastereomers to consist of a mixture of (SSSC) and (RSSC) diastereomers in the ratio
80:20. Deprotection of N-t-Boc-D-methionine sulfoxide obtained by biotransformation of N-t-Boc-D-
methionine using B. bassiana was performed in an exactly analogous experiment.

H. L. Holland et al. / Tetrahedron: Asymmetry 10 (1999) 2833–2843
2843
Acknowledgements
This work was funded by NSERC. We are grateful to Dr. C. S. Frampton of Roche Discovery Welwyn,
UK, for the determination of the X-ray crystallographic data, and to Mr. T. Jones of Brock University for
help with the acquisition of spectral data.
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