Highly enantioselective biohydrolysis of sec-alkyl sulfate esters with inversion of configuration catalysed by Pseudomonas spp.

Article abstract of DOI:10.1002/ejoc.200700637

In search of highly enantioselective microbial sec-alkyl sulfatase activity, a broad screening among bacteria, fungi and Archaea revealed several Ralstonia and Pseudomonas spp. as valuable sources, whereas fungi were completely inactive. In particular, Pseudomonas sp. DSM 6611 was able to hydrolyse the (R) enantiomers of a broad range of rac-sec-alkyl sulfate esters with excellent enantioselectivities (E > 200) to furnish the corresponding inverted (S)-sec-alcohols in high ee's. The substrate range of this organism was remarkably broad and bulky groups were also nicely tolerated. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700637

FULL PAPER
DOI: 10.1002/ejoc.200700637
Highly Enantioselective Biohydrolysis of sec-Alkyl Sulfate Esters with
Inversion of Configuration Catalysed by Pseudomonas spp.
[a]
[a]
Petra Gadler and Kurt Faber*
Keywords: Enzymes / Inversion of configuration / Enantioselectivity / Biohydrolysis / Biocatalysis
In search of highly enantioselective microbial sec-alkyl sulfat-
ase activity, a broad screening among bacteria, fungi and
Archaea revealed several Ralstonia and Pseudomonas spp.
as valuable sources, whereas fungi were completely inactive.
sulfate esters with excellent enantioselectivities (E Ͼ 200) to
furnish the corresponding inverted (S)-sec-alcohols in high
ee’s. The substrate range of this organism was remarkably
broad and bulky groups were also nicely tolerated.
In particular, Pseudomonas sp. DSM 6611 was able to hydro- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
lyse the (R) enantiomers of a broad range of rac-sec-alkyl
Germany, 2007)
Introduction
found to act predominantly through retention of configura-
tion on sulfated carbohydrates. This type of enzyme is
also present in humans (“human aryl sulfatase A” [EC
[
5]
Sulfatases are a heterogenic group of hydrolytic enzymes
that catalyse the cleavage of the sulfate ester bond to yield
the corresponding alcohol and hydrogen sulfate. Depending
on the mechanism of action of the enzyme, this reaction
may proceed either through retention or inversion of config-
uration at the chiral carbon centre (Scheme 1). Whereas
breakage of the S–O bond leads to retention, C–O bond
3
.1.6.8]) where it is involved in the degradation of glycos-
aminoglycans or in the synthesis of steroid hormones
“STS-steroid sulfatase” [EC 3.1.6.2]). Because of the fact
(
that various diseases are the result of (single or multiple)
[6]
sulfatase deficiency, this subclass of sulfatases is exten-
sively studied. Sequence analysis among aryl sulfatases led
to the identification of the highly conserved consensus
motif –C/S–X–P–X–R– among eukaryotic and prokaryotic
aryl sulfatases.
[
1]
cleavage results in inversion of configuration (Scheme 1).
The rare feature of double selectivities Ϫ stereoselectivity
in the context of inversion/retention and enantioselectivity
regarding the preference for a given substrate enantiomer
Ϫ makes them valuable candidates for the deracemisation
of sec-alcohols by enantioconvergent hydrolysis of their
corresponding sulfate esters.[
A more recent classification of sulfatases is based on
mechanistic aspects (rather than on substrate structure) and
[7]
defines them into three classes: (1) In eukaryotic aryl sul-
2,3]
fatases possessing the above-mentioned consensus motif, a
cysteine (or serine) residue within the active site is post-
translationally modified into an α-formylglycine moiety,
which is transformed into its corresponding aldehyde hy-
drate species. Being a powerful nucleophile, the latter at-
tacks the S atom thereby cleaving the S–O bond of the sul-
fate ester substrate; the absolute configuration at carbon
[
8]
II
is retained. (2) Class 2 sulfatases belong to the Fe α-
ketoglutarate-dependent dioxygenase superfamily and are
involved in the oxidative cleavage of a sulfate ester at the
expense of α-ketoglutarate into the corresponding aldehyde
Scheme 1. Stereochemical course of the biocatalytic hydrolysis of
sulfate esters by sulfatases.
[
9]
and inorganic sulfate. Because the latter occurs along with
the destruction of the chiral carbon centre, they are of little
use for stereoselective biotransformations. (3) Members of
the third class are related to metallo-β-lactamases contain-
ing two Zn2 atoms in the active site, which activate a water
molecule to form a (formal) hydroxide ion. The latter per-
forms nucleophilic attack on the substrate thereby yielding
the corresponding alcohol and inorganic sulfate. As a result
of the fact that only prim-sulfate esters were used as sub-
strates so far, the stereochemical features of this mechanism
According to their preferred substrate type, sulfatases
were previously classified into carbohydrate, aryl and alkyl
sulfatases. Carbohydrate and aryl sulfatases have been
[
4]
+
[
a] Department of Chemistry, Organic and Bioorganic Chemistry,
University of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
Fax: +43-316-380-9840
E-mail: Kurt.Faber@uni-graz.at
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2007, 5527–5530
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5527

P. Gadler, K. Faber
FULL PAPER
with respect to retention or inversion at the chiral carbon substrate tolerance was significantly wider and E values
are still unknown. In addition, the exact position of nucleo- were excellent (E Ͼ 200). Protein purification was not at-
[
7]
philic attack (at the sulfur or carbon atom) is still unclear.
tempted owing to difficult (extremophilic) cultivation con-
In contrast to sulfatases described above, much less is ditions (85–90 °C, pH 1–2) and insufficient yields in bio-
known about the mechanism of inverting sec-alkyl sulfat- mass. Because of the total lack of sequence information on
ases. The latter were first described during the 1970s in de- inverting alkyl sulfatases, genetic methods cannot be em-
tergent-degrading bacteria (Pseudomonas and Comamonas ployed.
spp.), which were isolated from sewage sludge.[ Subse-
10]
Aiming at a mesophilic microbial source for inverting
quent studies on these organisms revealed the presence of sec-alkyl sulfatases, which would yield sufficient biomass
three sec-alkyl sulfatases in Pseudomonas C12B NCIMB under standard conditions within short growth periods, we
11753, two of which (termed S1 and S2) were acting turned our attention to Pseudomonas spp. and closely re-
through inversion of configuration on rac-2-octyl sulfate al- lated strains.
beit with opposite enantiopreference,[ whereas the third
one (termed S3) was only active towards symmetrical long-
chain alkyl sulfates.[ Comamonas terrigena NCIMB 8193
11]
12]
Results and Conclusions
was shown to possess two inverting sec-alkyl sulfatases sim-
ilar to those of Pseudomonas C12B.[
13]
Interest in the
The screening for alkyl sulfatase activity among Pseu-
domonas spp. and related bacteria was guided by initial re-
[
12]
enantioselectivities of these enzymes arose rather slowly.
First hints on a certain enantiopreference arouse from the ports on the presence of alkyl sulfatases in Pseudomonas
chance observation that several rac-alkyl sulfates were C12B NCIMB 11753 and Comamonas terrigena NCIMB
[
10]
rapidly hydrolysed until 50% conversion, whereas further 8193. As a result of the occurrence of multiple alkyl sul-
reaction took place at a much slower rate.[ However, de- fatases,
14]
[11–14]
whole (resting) cells were not assumed to show
tailed studies were undertaken only two decades later: in reasonable enantioselectivities, but served as proof-of-prin-
search for alkyl sulfatase activities for the stereoselective hy- ciple. Phylogenetic selection of bacteria known to show a
drolysis of sec-alkyl sulfate esters, Rhodococcus ruber DSM broad secondary metabolism within the Pseudomonads,[
20]
[
15]
4
4541
Whole (resting) cells of this strain were found to hydrolyse stonia spp. as well as Rhodococcus-related actinomycetes,
R)-2-octyl sulfate into (S)-2-octanol. Although the stereo- led to a set of 88 bacterial strains, which were tested for
was identified as the first promising candidate. such as Comamonas, Xanthomonas, Xanthobacter and Ral-
(
[
16]
selectivity of sulfatase RS2 isolated from this strain was sec-alkyl sulfatase activity by using rac-2-octyl sulfate as a
excellent with regard to the strict inversion of configuration, test substrate.[21] This selection was complemented by 59
the enantioselectivity for (ω-1)-sec-alkyl sulfate esters Archaea and fungi, which are known to possess rich inor-
ranged from poor to a modest E value of 21 for rac-2-octyl ganic[22] and organic sulfur metabolism,
sulfate. Although these values could be enhanced to E Ͼ (Scheme 2, Table 1).
[23]
respectively
3
+
200 in the presence of Fe , the gain in selectivity was paid
A range of bacteroides exhibited low-to-excellent activi-
for by a loss in activity, which rendered this “enantioselec- ties, but poor enantioselectivities, which typically indicates
[
17]
tive-inhibition” method
unsuitable for preparative-scale the presence of multiple sulfatase enzymes competing for
[
18]
experiments. In addition, the substrate tolerance of RS2 the substrate (Table 1, Entries 1–6). Only slightly better
proved to be limited to linear sec-alkyl sulfate esters and its selectivities were observed with a range of Xanthobacter
biochemical characterisation was impeded by limited pro- spp. (Table 1, Entries 7–10, Emax = 7.6), and with Com-
[
16]
tein stability.
amonas sp. DSM 15091 the enantioselectivity approached a
Following the assumption that microorganisms pos- synthetically useful value of E = 16 (Table 1, Entry 11).
sessing strong inorganic sulfur metabolism with virtually all From two highly active Ralstonia spp., DSM 6428 showed
sulfur oxidation states from –2 to +6 might also be able an E value of 21 (Table 1, Entry 13). The clear hits of this
to metabolise sulfated organic species, a screening among screening process were found among the Pseudomonas spp.
Archaea was carried out. Among anaerobic Sulfolobus spp., (Table 1, Entries 14–16). Whereas NCIMB 11753, which is
Sulfolobus acidocaldarius DSM 639[ was identified to pos- known to express alkyl sulfatases S1 and S2 possessing op-
19]
[
11]
sess the desired inverting sec-alkyl sulfatase activity. The posite enantiopreference, exhibited only modest enantio-
Scheme 2. Enantioselective inverting biohydrolysis of sec-alkyl sulfate esters rac-1a–9a.
528
5
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Eur. J. Org. Chem. 2007, 5527–5530

Highly Enantioselective Biohydrolysis of sec-Alkyl Sulfate Esters
Table 1. Biohydrolysis of 2-octyl sulfate (rac-2a) by using (resting) monas sp. DSM 6978 showed excellent enantioselectivities
whole bacterial cells.
for long-chain linear substrates rac-2a–4a, albeit at moder-
Activity[a] Enantioselectivity
ate rates; again, steric hindrance prevented the hydrolysis of
bulky substrates rac-6a–9a. The clear hit from this study
was Pseudomonas sp. DSM 6611: with two exceptions, it
converted all of the substrates at modest-to-good rates with
almost absolute enantioselectivity (E Ͼ 200). Because steri-
cally demanding compounds bearing phenyl or cyclohexyl
groups were nicely accepted, steric hindrance did not seem
to play a measurable role. Overall, all strains showed a more
or less pronounced enantiopreference for the (R) enantio-
mer by acting with strict inversion of configuration.
Detailed biochemical characterisation of the sec-alkyl
sulfatase from Pseudomonas sp. DSM 6611 is currently on-
going with the aim to elucidate its unique catalytic mecha-
nism and its application in the deracemisation of sec-
alcohols by enantioconvergent hydrolysis of their corre-
sponding racemic sulfate esters.
Entry Strain
E
1
2
3
4
5
6
Bacillus sphaericus FCC 098
Achromobacter sp. FCC 175
Nocardia nova DSM 43843
Rhizobiaceae sp. FCC 099
Cupriavidus necator DSM 5536
Gulosibacter molinativorax DSM 13485
Ϯ
Ϯ
1.1
2.0
2.0
2.0
4.6
5.3
++
++
Ϯ
++
7
8
9
Xanthobacter autotrophicus DSM 431
Xanthobacter sp. DSM 6696
Xanthobacter flavus DSM 338
++
Ϯ
2.0
2.3
2.7
7.6
Ϯ
10
Xanthobacter autotrophicus DSM 3874
Ϯ
11
Comamonas sp. DSM 15091
Ϯ
16
1
13
2
Ralstonia eutropha SPT0001 FCC120
Ralstonia sp. DSM 6428
++
++
1.0
21
1
4
Pseudomonas sp. NCIMB 11753
Pseudomonas sp. DSM 6978
Pseudomonas sp. DSM 6611
Ϯ
Ϯ
13
Ͼ200
Ͼ200
1
5
16
++
Experimental Section
[
a] Activity range denoted as:Ϯ= low (conversion Ͻ5%), + = moder-
ate (conversion 5–10%), ++ = high (conversion Ͼ 10%) within 24 h
Synthesis of Alkyl Sulfate Esters: Sulfate esters rac-1a–9a and (R)-
under standard conditions.
and (S)-2a were prepared from the corresponding alcohols by using
3 3
NEt ·SO
according to a known procedure.[ Sulfate esters were
24]
obtained as lyophilised powders, and their NMR spectra were in
agreement with the reported values.
selectivity (E = 13), DSM 6978 and DSM 6611 hydrolysed
the (R) enantiomer from rac-2 with excellent enantio-
selectivity and with inversion of configuration to furnish
inverted sec-alcohol (S)-2b (E Ͼ 200). The latter was proven
by the fact that the biohydrolysis of enantiomerically pure
[24]
Screening for Sulfatase Activity: Lyophilised whole cells (30–50 mg)
were rehydrated in Tris·HCl buffer (500–600 µL, pH 7.5, 100 m)
for 1 h at 30 °C whilst shaking at 120 rpm. Then, an aliquot
–1
(
200 µL) from a substrate stock solution (20 mgmL rac-2a) was
(R)-2-octyl sulfate solely gave (S)-2-octanol, whereas (S)-2a
added as a test substrate. Other substrates were used in the concen-
–
1
was not converted at all.
tration of 50 mgmL . The reaction mixture was incubated at 30 °C
These encouraging results prompted us to investigate the whilst shaking at 120 rpm for 24 h. The samples were extracted
substrate-selectivity pattern of Pseudomonas spp. in more with ethyl acetate (600 µL) and centrifuged at 13.000 rpm for 2 min
to separate the organic layer from the cell/buffer suspension. The
detail (Table 2).
2 4
organic layer was dried with Na SO and 100 µL of an internal
The lead organism Pseudomonas NCIMB 11753 (pre-
viously denoted C12B) was able to hydrolyse linear (ω-1)-
sulfate esters rac-1a–3a at low rates and with modest
enantioselectivities (Emax = 13 for rac-2a). Moving the reac-
tive group towards the centre of the molecule (rac-4a, rac-
–
1
standard (10 mgmL of rac-2-dodecanol) was added. Conversions
were measured with an achiral GC column and values were calcu-
lated from calibration curves. Afterwards, positive hits were deriv-
atised overnight with acetic anhydride (60 µL) and catalytic DMAP
for chiral analysis. The reaction was quenched with tap water
5
a) led to a gradual decrease in selectivities due to the in-
creasing spatial similarity of side chains. Bulky substituents was treated as described above. For details on the GC analyses, see
rac-6a–9a) were not tolerated at all. In contrast, Pseudo- the Supporting Information.
(
300 µL), centrifuged for 2 min at 13.000 rpm and the organic layer
(
Table 2. Substrate tolerance and enantioselectivities of Pseudomonas spp. NCIMB 11753, DSM 6978 and DSM 6611.
Substrate
Pseudomonas sp. NCIMB 11753
Pseudomonas sp. DSM 6978
Pseudomonas sp. DSM 6611
Conversion
%]
Enantioselectivity
Conversion
[%]
Enantioselectivity
Conversion
[%]
Enantioselectivity
[
E
E
E
rac-1a
rac-2a
rac-3a
rac-4a
rac-5a
rac-6a
rac-7a
rac-8a
rac-9a
8
4
2
2
10
8
n.c.
n.c.
n.c.
4
13
2
8
5
1
7
4
2
2
n.c.
n.c.
n.c.
n.c.
n.d.[a]
Ͼ200
Ͼ200
Ͼ200
4
17
21
7
18
20
9
n.c.
15
5
Ͼ200
Ͼ200
Ͼ200
Ͼ200
6
n.d.[
a]
Ͼ200
1.2
n.d.[
a]
n.d.
[a]
n.d.
[a]
[
a]
[a]
n.d.
n.d.
Ͼ200
Ͼ200
n.d.[
a]
n.d.
[a]
[a] n.d. = Not determined due to low (or no) conversion.
Eur. J. Org. Chem. 2007, 5527–5530
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5529

P. Gadler, K. Faber
FULL PAPER
Determination of Stereochemical Pathway and Absolute Configura-
tion: Stereopreferences were determined by using enantiopure (R)-
and (S)-2-octyl sulfate (2a) as the substrate and prepared as de-
scribed above; absolute configurations of alcohols 1b–9b were elu-
cidated by chiral GC by coinjection with commercially available or
independently synthesised reference materials.
[8] I. Boltes, H. Czapinska, A. Kahnert, R. von Buelow, T. Dierks,
B. Schmidt, K. von Figura, M. A. Kertesz, I. Uson, Structure
2
001, 9, 483–491.
[
9] I. Mueller, A. Kahnert, T. Pape, G. M. Sheldrick, W. Meyer-
Klaucke, T. Dierks, M. A. Kertesz, I. Uson, Biochemistry 2004,
4
3, 3075–3088.
[
10] K. S. Dodgson, G. F. White, J. W. Fitzgerald, Sulfatases of Mi-
crobial Origin, CRC Press, Boca Raton, 1982, vols. 1 and 2.
[11] B. Bartholomew, K. S. Dodgson, G. W. J. Matcham, D. J.
Supporting Information (see footnote on the first page of this arti-
cle): Source of organisms, culture conditions and GC analyses of
sec-alcohols 1b–9b.
Shaw, G. F. White, Biochem. J. 1977, 165, 575–580.
[
12] D. J. Shaw, K. S. Dodgson, G. F. White, Biochem. J. 1980, 187,
181–196.
[
[
13] J. W. Fitzgerald, Biochem. J. 1975, 149, 477–480.
14] G. W. J. Matcham, B. Bartholomew, K. S. Dodgson, J. W. Fitz-
gerald, W. J. Payne, FEMS Microbiol. Lett. 1977, 1, 197–200.
15] M. Pogorevc, W. Kroutil, S. R. Wallner, K. Faber, Angew.
Chem. Int. Ed. 2002, 41, 4052–4054.
Acknowledgments
[
[
[
We would like to thank K.-H. Engesser, G. Guebitz and their co-
workers for the generous donation of strains and their valuable
support in microbiology. Financial support by the Austrian Science
Fund (FWF, project no. P18689) is gratefully acknowledged.
16] M. Pogorevc, K. Faber, Appl. Environ. Microbiol. 2003, 69,
2810–2815.
17] a) F. Theil, Tetrahedron 2000, 56, 2905–2919; b) Z.-W. Guo,
C. J. Sih, J. Am. Chem. Soc. 1989, 111, 6836–6841; c) R. Am-
mon, H. Fischgold, Biochem. Z. 1931, 234, 54–61.
18] M. Pogorevc, U. T. Strauss, T. Riermeier, K. Faber, Tetrahe-
dron: Asymmetry 2002, 13, 1443–1447.
[
[1] P. Gadler, K. Faber, Trends Biotechnol. 2007, 25, 83–88.
[
19] S. R. Wallner, B. M. Nestl, K. Faber, Org. Lett. 2004, 6, 5009;
S. R. Wallner, B. M. Nestl, K. Faber, Org. Biomol. Chem. 2005,
[2] S. R. Wallner, M. Pogorevc, H. Trauthwein, K. Faber, Eng. Life
Sci. 2004, 4, 512–516.
3, 2652.
[
3] a) K. Faber, Chem. Eur. J. 2001, 7, 5004–5010; b) P. Gadler,
S. M. Glueck, W. Kroutil, B. M. Nestl, B. Larissegger-Schnell,
B. T. Ueberbacher, S. R. Wallner, K. Faber, Biochem. Soc.
Trans. 2006, 34, 296–300.
[
20] N. J. Palleroni, R. Kunisawa, R. Contopoulou, M. Doudoroff,
Int. J. Syst. Bacteriol. 1973, 23, 333–339.
[21] For a complete list of inactive strains see the Supporting Infor-
mation.
[
[
4] M. A. Kertesz, FEMS Microbiol. Rev. 1999, 24, 135–175.
5] In the majority of biochemical studies, the stereochemical
course of the biohydrolysis (inversion vs. retention) cannot be
answered, as nonchiral p-nitrophenyl sulfate was used as the
substrate.
[22] a) H. Huber, K. O. Stetter, J. Biotechnol. 1998, 64, 39–52; b) C.
Bertoldo, C. Dock, G. Antranikian, Eng. Life Sci. 2004, 4, 521–
532.
[23] Besides sulfate esters, sulfonates are widespread in nature and
make up over 95% of the sulfur content of most aerobic soils,
see ref.[
4]
[6] S. R. Hanson, M. D. Best, C.-H. Wong, Angew. Chem. Int. Ed.
2
004, 43, 5736–6763.
[24] M. Pogorevc, K. Faber, Tetrahedron: Asymmetry 2002, 13,
[
7] G. Hagelueken, T. M. Adams, L. Wiehlmann, U. Widow, H.
Kolmar, B. Tuemmler, D. W. Heinz, W.-D. Schubert, Proc.
Natl. Acad. Sci. USA 2006, 103, 7631–7636.
1435–1441.
Received: July 11, 2007
Published Online: September 21, 2007
5530
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Eur. J. Org. Chem. 2007, 5527–5530