A highly enantioselective biocatalytic sulfoxidation by the topsoil bacterium Pseudomonas frederiksbergensis

Article abstract of DOI:10.1016/j.tetasy.2003.12.030

The biocatalytic oxidation of various organic sulfides with whole cells of the commercially available topsoil bacterium Pseudomonas frederiksbergensis DSM 13022 affords the corresponding (S)-sulfoxides in high (up to >99% ee) enantiomeric excess.

Full text of DOI:10.1016/j.tetasy.2003.12.030

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 983–985
A highly enantioselective biocatalytic sulfoxidation by the topsoil
bacterium Pseudomonas frederiksbergensis
b
a
b
Waldemar Adam,a, Frank Heckel, Chantu R. Saha-Moller, Marcus Taupp and
*
€
Peter Schreierb,*
aInstitute of Organic Chemistry, University of W€urzburg, Am Hubland, 97074 W€urzburg, Germany
bInstitute of Food Chemistry, University of W€urzburg, Am Hubland, 97074 W€urzburg, Germany
Received 12 December 2003; accepted 23 December 2003
Abstract—The biocatalytic oxidation of various organic sulfides with whole cells of the commercially available topsoil bacterium
Pseudomonas frederiksbergensis DSM 13022 affords the corresponding (S)-sulfoxides in high (up to >99% ee) enantiomeric excess.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
characterized. DNA–DNA hybridization data showed
that this strain JAJ28 (DSM 13022) belongs to a new
species, which was named P. frederiksbergensis sp.
Nov.15 Although, this bacterial strain is known since
1996,14 biocatalytic applications of this microorganism
are essentially unknown. To date, only the degradation
of phenanthrene by this topsoil bacterium has been
carried out.14 Herein, we report on the very first appli-
cation of P. frederiksbergensis in asymmetric oxidation,
in particular, the enantioselective oxidation of alkyl aryl
sulfides (Scheme 1).
Enantiomerically pure sulfoxides play an important role
in asymmetric synthesis either as chiral building blocks
or stereodirecting groups.1 Recently, metal- and
enzyme-catalyzed asymmetric sulfoxidations have been
developed for the preparation of optically active sulf-
oxides.2;3 Enzymes, especially peroxidases, catalyze effi-
ciently the enantioselective sulfoxidation of a variety of
sulfides.4–6 Despite this success, asymmetric transfor-
mations with isolated enzymes are tedious and expen-
sive, a major disadvantage for preparative applications.7
In contrast, whole-cell systems (fungi and bacteria) are
available as biocatalysts in sufficient quantities for pre-
parative purposes through self-replication. Thus, several
bacterial strains have been successfully employed for the
biocatalytic sulfoxidation.8–10 These studies revealed
that intact-cell bacterial oxidation is very promising for
the development of environmentally benign, efficient
asymmetric sulfoxidation. Therefore, in continuation of
our investigations on the biocatalytic oxidation with
bacteria,11–13 we have searched for new bacterial strains
with broad scope of substrate tolerance for the enan-
tioselective oxidation of sulfides.
O
P. frederiksbergensis
S
*
S
1
2
1
2
R
R
R
R
30 ˚C, 120 rpm, 18 h
1
(S)-2
Scheme 1. Enantioselective sulfoxidation by the topsoil bacterium
P. frederiksbergensis.
2. Results and discussion
To assess optimum reaction conditions for the asym-
metric sulfoxidation by P. frederiksbergensis, screening
experiments were carried out with methyl phenyl sulfide
1a as the model substrate. For this purpose, sulfide 1a
was treated with pregrown bacteria (for details see
Experimental) for particular periods of time and, sub-
sequently, the yield and ee value of methyl phenyl sulf-
oxide were determined (Fig. 1). As shown in Figure 1,
the yield of the sulfoxide increased gradually for reac-
tion times of up to 70 h, while high enantioselectivity
Recently, a topsoil bacterium was isolated as a Pseu-
domonas biovar at a coal gasification site in Frederiks-
berg, Copenhagen, Denmark,14 a site propitious for the
occurrence of elemental sulfur and its organic deriva-
tives. The strain was phenotypically and genotypically
* Corresponding authors. Tel.: +49-931-8885480; fax: +49-931-8885-
484; e-mail: schreier@pzlc.uni-wuerzburg.de
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.12.030

984
W. Adam et al. / Tetrahedron: Asymmetry 15 (2004) 983–985
the topsoil bacterium P. frederiksbergensis was achieved.
100
95
90
85
80
75
70
To assess the effect of the alkyl side chain on the
enantioselectivity, the n-propyl derivative 1e was treated
with the microorganism. As shown in entry 5, signifi-
cantly lower enantioselectivity (81% ee of 2e) was
observed compared to that for the methyl derivative 1a
(91% ee, entry 1); thus, the chain length has a pro-
nounced effect on the enantioselectivity of sulfoxidation
by this bacterium. Moreover, a branched alkyl chain
dramatically lowers the substrate acceptance of
P. frederiksbergensis; for example, the isopropyl deriv-
ative 1f was barely consumed by this topsoil bacterium
(entry 6). Not only alkyl aryl sulfides 1a–e, but also
dialkyl sulfides such as cyclohexyl methyl sulfide 1g are
sulfoxidized in good enantioselectivity (70% ee, entry 7).
In contrast, the benzyl methyl sulfide 1h was not ac-
cepted by P. frederiksbergensis (entry 8).
6
12
24
48
67
70
73
76
91
97
time [h]
Figure 1. Time profile of the oxidation of methyl phenyl sulfide (1a)
with pregrown P. frederiksbergensis; conversion (–d–) of methyl
phenyl sulfide and enantiomeric excess (–j–) of methyl phenyl sulf-
oxide.
For all enantioenriched sulfoxides described herein, the
S enantiomer predominates and in no case was the
corresponding sulfone formed in these bacterial trans-
formations. The observed sense of the enantioselectivity
in the sulfoxidation by P. frederiksbergensis is opposite
to that of peroxidases, for example, chloroperoxidase.16
(>90% ee) was achieved already after 12 h of conversion.
After about 18 h, the enantioselectivity did not increase
any more for the model sulfide 1a and, therefore, this
reaction time was applied for the oxidation of the other
sulfide substrates. To assess the appropriate pH value in
the bacterial sulfoxidation, a pH profile was run for the
sulfoxidation of the methyl phenyl sulfide 1a by
P. frederiksbergensis. Essentially enantiopure sulfoxide
was obtained at the broad pH range of 6–8 (data not
shown). Thus, an adjusted Dworkin mineral nutriment
solution of neutral pH value (pH ¼ 7) was used as the
basic medium for the bacterial sulfoxidation. A 50%
aqueous solution of glucose was chosen as the carbon
source and optimal bacterial growth was achieved by
using 15–30 g glucose per liter of growth medium.
3. Conclusions
In summary, we have described for the first time the
enantioselective sulfoxidation of a series of organic
sulfides by P. frederiksbergensis, whose biocatalytic
potential has so far been hardly explored for oxidation
purposes. These encouraging results should stimulate
further work on this readily available soil bacterium,
such as the identification of its oxidation enzyme and the
utilization of this microorganism for large-scale appli-
cation in sulfoxidation and other oxidative transfor-
mations.
The optimized conditions were applied in the biocata-
lytic oxidation of a broad series of sulfides with the soil
bacterium P. frederiksbergensis (Table 1). The yields of
the sulfoxides range between 4% and 48% (data not
shown in Table 1), but have as yet not been optimized,
since the emphasis of this work lies on the enantio-
selectivity of the sulfoxidation. The standard model
substrate, methyl phenyl sulfide 1a, was sulfoxidized in
an enantiomeric excess of 91% (entry 1). In the case of
para substitution, as in the sulfides 1b–d (entries 2–4),
essentially enantiopure sulfoxides were obtained. Inter-
estingly, the enantioselectivity is independent of the
electronic properties of the para substituents, since for
both electron-donating (Me, MeO) and electron-with-
drawing (Cl) groups excellent enantiodifferentiation by
4. Experimental
4.1. General procedure for the biotransformation of
sulfides by P. frederiksbergensis
The P. frederiksbergensis strain (DSM13022) was
€
obtained from the DSMZ (Deutsche Sammlung fur
Table 1. Enantioselective oxidation of sulfides 1 to sulfoxides 2 by P. frederiksbergensis
Entry
Substrate
R1
R2
Conversion (%)
(S)-2 ee (%)a
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
Ph
Me
Me
Me
Me
n-Pr
i-Pr
Me
Me
87
100
98
91
>99
>99
96
4-Me–Ph
4-MeO–Ph
4-Cl–Ph
Ph
86
27
81
Ph
c-Hex
Ph–CH2
<5
98
––
1g
1h
70
<5
––
a Enantiomeric excess determined by a multidimensional gas-chromatography/mass spectrometry combination on a 2,3-diethyl-6-tert-butyldimeth-
ylsilyl-b-cyclodextrin column, error 1.5% of the stated value; the S absolute configuration was assessed from CPO data (Ref. 16).

W. Adam et al. / Tetrahedron: Asymmetry 15 (2004) 983–985
985
Mikroorganismen und Zellkulturen, Braunschweig,
Germany) and maintained at 4 °C on Standard Methods
References and notes
1. Mata, E. G. Phosphorus, Sulfur Silicon Relat. Elem. 1996,
117, 231–286.
ꢀ
Agar (from Bio Merieux, Marcy lÕEtoile, France). To
elucidate the scope of sulfoxidation activity by P. fred-
eriksbergensis, several organic sulfides [analytical grade
obtained from Fluka (Seelze, Germany), Sigma-Aldrich
(Taufkirchen, Germany), or Lancaster (Morecambe,
England)] were used for biotransformation. The bacte-
rial sulfoxidation experiments were carried out in 300-
mL Erlenmeyer flasks, which were charged with 100 mL
Dworkin mineral nutriment solution17 and 375 lL
Pseudomonas trace element solution.18 This medium was
sterilized at 121 °C for 16 min prior to the addition of
750 lL 50% steril glucose solution (standard conditions).
After inoculation with bacterial material, the culture was
pregrown for 24 h and then shaken in the presence of
100 lmol of the appropriate sulfide for 18 h at 30 °C with
120 rpm. Subsequently, the bacterial cells were treated by
ultrasound to release the sulfoxides that are retained in
the cells. After centrifugation (9000 rpm, 15 min), the
bacterial suspension was successively extracted twice
with 100 mL ethyl ether and 100 mL dichloromethane
(all solvents were distilled before use). Finally, the
products were submitted to gas-chromatographic anal-
ysis and mass spectrometry (GC–MS). The chiral anal-
€
2. Adam, W.; Lazarus, M.; Saha-Moller, C. R.; Weichold,
O.; Hoch, U.; Haring, D.; Schreier, P. Adv. Biochem. Eng.
Biotechnol. 1999, 63, 73–108.
3. Colonna, S.; del Sordo, S.; Gaggero, N.; Carres, G.; Pasta,
P. Heteroat. Chem. 2002, 13, 467–473.
4. Colonna, S.; Gaggero, N.; Casella, L.; Carrea, G.; Pasta,
P. Tetrahedron: Asymmetry 1992, 3, 95–106.
5. van Deurzen, M. P. J.; Remkes, I. J.; van Rantwijk, F.;
Sheldon, R. A. J. Mol. Catal. A: Chem. 1997, 117, 329–337.
€
€
6. Adam, W.; Mock-Knoblauch, C.; Saha-Moller, C. R.
J. Org. Chem. 1999, 64, 4834–4839.
€
7. Adam, W.; Heckel, F.; Saha-Moller, C. R.; Schreier, P.
J. Organomet. Chem. 2002, 661, 17–29.
8. Holland, H. L. Chem. Rev. 1988, 88, 473–485.
9. Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.;
Wright, M. A. Tetrahedron: Asymmetry 1996, 7, 365–368.
10. Boyd, D. R.; Sharma, N. D.; Haughey, S. A.; Kennedy,
M. A.; McMurray, B. T.; Sheldrake, G. N.; Allen, C. C.
R.; Dalton, H.; Sproule, K. J. Chem. Soc., Perkin Trans. 1
1998, 1929–1933.
€
11. Adam, W.; Lukacs, Z.; Kahle, C.; Saha-Moller, C. R.;
Schreier, P. J. Mol. Catal. B: Enzym. 2001, 11, 377–385.
€
12. Adam, W.; Lukacs, Z.; Harmsen, D.; Saha-Moller, C. R.;
Schreier, P. J. Org. Chem. 2000, 65, 878–882.
€
ysis of these products was performed by
a
13. Adam, W.; Lukacs, Z.; Saha-Moller, C. R.; Schreier, P.
multidimensional GC–MS combination on a 2,3-diethyl-
6-t-butyldimethylsilyl-b-cyclodextrin column. For GC
analysis, Thermo Finnigan Gaschromatographs 8000
series were utilized and for chiral analysis a moving
column switching system (MCSS) was used for peak
transfer. High-resolution mass spectra were obtained on
a Finnigan MD800. The absolute configurations of the
sulfoxides were determined by comparison with
authentic samples (prepared by enzymatic sulfoxidation
with chloroperoxidase (CPO) according to the previ-
ously reported procedure4) and literature data.16
J. Am. Chem. Soc. 2000, 122, 4887–4892.
14. Johnsen, K.; Andersen, S.; Jacobsen, C. S. Appl. Environ.
Microbiol. 1996, 62, 3818–3825.
15. Andersen, S. M.; Johnsen, K.; Sørensen, J.; Nielsen, P.;
Jacobsen, C. S. Int. J. Syst. Evolut. Microbiol. 2000, 50,
1957–1964.
16. Colonna, S.; Gaggero, N.; Manfredi, A.; Casella, L.;
Gullotti, M.; Carrea, G.; Pasta, P. Biochemistry 1990, 29,
10465–10468.
17. Dworkin, M.; Forster, J. W. J. Bact. 1958, 75, 592–603.
18. Atlas, R. M. In Handbook of Microbiological Media; Parks,
L. C., Ed.; 2nd ed.; CRC: Boca Raton, 1993; p 1.