Highly enantioselective stereo-inverting sec-alkylsulfatase activity of hyperthermophilic Archaea

Article abstract of DOI:10.1039/b504883d

rac-sec-Alkyl sulfate esters 1a-8a were resolved in low to excellent enantioselectivities with E-values up to > 200 using whole cells of aerobically-grown hyperthermophilic sulfur-metabolizers, such as Sulfolobus solfataricus DSM 1617, Sulfolobus shibatae DSM 5389 and, most notably, Sulfolobus acidocaldarius DSM 639. Significantly enhanced selectivities were obtained using cells grown on sucrose-enriched Brock-medium. The stereochemical course of this biohydrolysis was shown to proceed with strict inversion of configuration, thus the preferred (R)-enantiomers were converted into the corresponding (S)-sec-alcohols to furnish a homochiral product mixture. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b504883d

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A R T I C L E
Highly enantioselective stereo-inverting sec-alkylsulfatase activity of
hyperthermophilic Archaea†
Sabine R. Wallner,a,b Bettina M. Nestla and Kurt Faber*a
a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz,
Heinrichstrasse 28, A-8010 Graz, Austria. E-mail: Kurt.Faber@Uni-Graz.at;
Fax: +43-316-380-9840; Tel: +43-316-380-5332
b Research Centre Applied Biocatalysis, Graz, Austria
Received 11th April 2005, Accepted 24th May 2005
First published as an Advance Article on the web 21st June 2005
rac-sec-Alkyl sulfate esters 1a–8a were resolved in low to excellent enantioselectivities with E-values up to >200 using
whole cells of aerobically-grown hyperthermophilic sulfur-metabolizers, such as Sulfolobus solfataricus DSM 1617,
Sulfolobus shibatae DSM 5389 and, most notably, Sulfolobus acidocaldarius DSM 639. Significantly enhanced
selectivities were obtained using cells grown on sucrose-enriched Brock-medium. The stereochemical course of this
biohydrolysis was shown to proceed with strict inversion of configuration, thus the preferred (R)-enantiomers were
converted into the corresponding (S)-sec-alcohols to furnish a homochiral product mixture.
which were shown to act on sec-alkyl sulfate esters by breaking
Introduction
the C–O bond of the sulfate ester, going in hand with inversion
Driven by the demand to improve the economic balance of
of configuration. As a result, a homochiral product mixture is
chemical processes for the synthesis of chiral compounds, the
obtained from racemic starting material. In order to remove
transformation of racemates into a single stereoisomeric product
the sulfate moiety from the remaining non-reacted sulfate ester
in quantitative yield has become a prime issue for contemporary
enantiomer, a protocol for acid-catalyzed hydrolysis with strict
asymmetric synthesis. Besides the widely employed dynamic
retention of configuration has been recently developed.17
kinetic resolution,1 enantio-convergent processes – during which
Based on vague hints on the stereospecific and enantiose-
each enantiomer is transformed into the same stereoisomeric
lective hydrolysis of alkyl sulfate esters,14 we recently reported
product via independent pathways, i.e. through retention and
an inverting alkylsulfatase (termed ‘RS2’) from Rhodococcus
inversion of configuration – were shown to be a promising
ruber DSM 44541.16,18 On the one hand, the enzyme displayed
alternative.2 Biocatalysts, which show this stereochemically
complex potential to affect the stereochemistry of the substrate
absolute stereospecificity by acting with strict inversion of con-
figuration on simple sec-alkyl esters; on the other hand, its enan-
tioselectivity was less than perfect: although 2-octyl sulfate (rac-
1a) was resolved with an acceptable E-value of 21, no appreciable
in a controlled fashion during catalysis are rather rare; they
encompass haloalkane dehalogenases,3 epoxide hydrolases4 and
sulfatases.5,6
enantioselectivities were observed for 3- and 4-octyl sulfate (E <
Sulfatases catalyze the hydrolytic cleavage of the sulfate ester
5).16 Attempts to enhance selectivities by enzyme inhibition (e.g.
bond. In contrast to the majority of hydrolytic biotransforma-
addition of Fe3+)19 were successful but (as usual in inhibition) led
tions catalyzed by lipases, esterases and proteases,7 which do
to a significant loss of catalytic activities. Furthermore, the sub-
not alter the stereochemistry of the substrate during catalysis,
strate tolerance of sulfatase RS2 was rather narrow, as substrates
the stereochemical course of sulfate ester hydrolysis can be
bearing bulky aryl groups (e.g. rac-4a) were not accepted.16
controlled by the choice of the appropriate subtype of sulfatase
Our search for novel (and more selective) inverting alkylsul-
enzyme (Scheme 1). On the one hand, arylsulfatases8 generally
fatases was led by the notion that organisms known to possess a
act through retention of configuration at the sulfated carbon
rich inorganic sulfur metabolism (encompassing inorganic sulfur
atom by cleavage of the S–O bond.9 Their mechanism of action
species of all oxidation states ranging from sulfide to sulfate20)
is well understood10,11 and comprises the nucleophilic attack of
might also act on sulfated organic species, such as alkyl sulfates.21
an aldehyde hydrate (formed from a Cys- or Ser-residue by post-
Sulfur-based redox-chains were a prime energy-source for life
translational modification12) onto the sulfur atom, by liberating
before molecular oxygen was released into the atmosphere by
the corresponding alcohol by retaining its stereochemistry.13 On
cyanobacteria some 3.5 billion years ago.22 Typically, sulfur-
the contrary, much less is known on inverting sulfatases,6,14–16
metabolizers belong to the kingdom of Archaea and were
adapted to the harsh environment of the early biosphere, such
as high growth temperatures (55–95 ◦C), low pH (0.0–4.0)
† Electronic supplementary information (ESI) available: general ana-
lytical and synthetic methods, the synthesis of substrates (rac-1a–9a)
and reference materials [(S)-3b, (S)-7d, (S)-8d, (S)-7e, (S)-8e, (S)-7f,
(S)-8f, (R)-7b, (R)-8b] and their spectroscopic data. See http://www.rsc.
org/suppdata/ob/b5/b504883d/
and/or high salinity (0.4–3.5 M NaCl).23 In their mode to gain
energy they are extremely flexible and exhibit a great variety
of pathways: obligate chemolithoautotrophs utilize only CO2,
hydrogen and different inorganic sulfur compounds, such as S2−,
Scheme 1 Enzymatic stereo-divergent hydrolysis of sulfate esters catalyzed by sulfatases.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 2 – 2 6 5 6 T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
2 6 5 2
©

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0
S22−, S8 , SO32−, S2O32−, SO42−, or nitrate. On the other hand,
grown strains could hydrolyze rac-1a (Table 1, entries 1–11).
Best reaction rates were obtained with three Sulfolobus spp.:
S. solfataricus DSM 1617, S. shibatae DSM 5389 and S.
acidocaldarius DSM 639 (entries 9–11). For the sterically more
demanding substrate rac-6a, only the latter strains exhibited
reasonable activities (entries 12–14). This trend was even more
pronounced for the branched long-chain substrate rac-9a, which
was only accepted at a slow rate by Sulfolobus acidocaldarius
DSM 639 (entry 15), which clearly emerged as the ‘champion’
with respect to reaction rates and substrate tolerance.28
chemo-organotrophs obtain their energy from the oxidation of
organic compounds (e.g. organic acids, alcohols, sugars, amino
0
2−
acids or polymers, such as starch or chitin) using S8 , SO4 or
molecular oxygen as the oxidant.24
Results and discussion
In order to cover a broad range of the kingdom of Archaea,25
a selection of extremophilic sulfur-metabolizers was chosen,
and these were grown aerobically26 and anaerobically27 on the
recommended media. Whole cells were tested for alkylsulfatase
activity at pH 2–3, which corresponds to that of their natural
habitats, using 2-octyl sulfate (rac-1a), 1-phenyl-2-propyl sulfate
(rac-6a) and 6-methylhept-5-ene-2-yl sulfate (‘sulcatyl sulfate’,
rac-9a) as substrates (Scheme 2). For all compounds, blank
experiments were performed in the absence of biocatalyst to
exclude any undesired spontaneous hydrolysis. From the begin-
ning, a clear picture emerged as all of the eight anaerobically-
grown strains27 proved to be completely inactive on the above-
mentioned substrates. On the contrary, all of the 11 aerobically-
A first look into the enantioselectivities – expressed as E-
values29 – by measuring the enantiomeric excess of the formed
sec-alcohol and the conversion using an internal standard
was promising (Table 1). Although no measurable enantio-
preference could be detected for the majority of strains showing
low activity (entries 1–8), the three most active Sulfolobus
spp. gave good to excellent enantioselectivities for rac-1a, with
Sulfolobus acidocaldarius DSM 639 again being best (E-value
106). Low values were observed for rac-6a (E-values up to 2.8).
In comparison to the majority of bacteria and fungi most
widely used for whole-cell biotransformations, the growth rates
of Archaea on the usually recommended complex media are
comparably low and values for optical densities did not exceed
an OD of ca. 0.15 after a period of 35 d, which is unacceptable
for preparative-scale experiments. In order to speed up the
growth rates, experiments were undertaken with respect to
medium optimisation for the most promising strain – Sulfolobus
acidocaldarius DSM 639 – by supplementing the basal medium
according to Brock et al.30 with various ‘simple’ carbon sources.
As shown in Fig. 1, a low cell density (OD 0.17) was obtained
on the standard Brock-medium after 35 d. Enrichment of this
medium by the addition of glucose at concentrations of 5, 10
and 20 g L−1 did not lead to any significant improvement in
growth rates. Similarly disappointing results were observed with
glycerol (20 g L−1) and mannitol (20 g L−1). Finally, a three-fold
improved growth rate was achieved by using sucrose (20 g L−1)
as a supplement, which led to an acceptable OD40d of 0.95.
With a greatly facilitated access to biomass in hand, enantio-
selectivities were re-checked for Sulfolobus acidocaldarius DSM
639 grown on sucrose-supplemented medium using substrates
rac-1a–9a (Table 2).
We were suprised to see that S. acidocaldarius DSM 639 grown
on sucrose-enriched medium showed significantly enhanced
enantioselectivitites: excellent enantioselectivity was observed
for 2-octyl sulfate (rac-1a, E > 200). The most plausible
explanation for this phenomenon is the selective induction of
an appropriate alkylsulfatase or the suppression of competing
enzyme(s) by the sucrose-enriched medium. In comparison,
Scheme 2 Enantioselective microbial hydrolysis of sec-alkyl sulfate
esters with inversion of configuration.
Table 1 Screening for sec-alkylsulfatase activity in aerobically-grown hyperthermophilic Archaeaa
Entry Substrate Microorganism
Growth medium
Yieldb (%) Product/ee (%) Enantioselectivity (E-value)
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
Acidianus brierley DSM 1651
Brock + sucrose + S8
Brock + sucrose + S8
Brock + sucrose + S8
Brock + sucrose + S8
Brock + sucrose + S8
Brock + sucrose + S8
Brock + sucrose + S8
7.4
8.0
8.4
8.3
25
13
9
11
56
42
33
(R)-1b/2
(R)-1b/2
(R)-1b/2
(S)-1b/2
(R)-1b/3
(R)-1b/3
(R)-1b/3
(R)-1b/3
(S)-1b/77
(S)-1b/92
(S)-1b/97
ca. 1
ca. 1
ca. 1
ca. 1
ca. 1
ca. 1
ca. 1
ca. 1
35
Sulfolobus metallicus DSM 6482
Sulfololobus hakoniensis DSM 7519
Sulfolobus acidocaldarius IFO 15159
Acidianus infernus DSM 3191
Acidianus ambivalens DSM 3772
Metallosphaera sedula DSM 5348
Sulfurisphaera ohwakuensis DSM 12421 Brock + sucrose + S8
Sulfolobus solfataricus DSM 1617
Sulfolobus shibatae DSM 5389
Sulfolobus acidocaldarius DSM 639
DSM #182
DSM #88
DSM #88
10
11
48
106
12
13
14
rac-6a
rac-6a
rac-6a
Sulfolobus solfataricus DSM 1617
Sulfolobus shibatae DSM 5389
Sulfolobus acidocaldarius DSM 639
DSM #182
DSM #88
DSM #88
20
20
16
(S)-6b/35
(S)-6b/43
(S)-6b/25
2.3
2.8
1.8
15
rac-9a
Sulfolobus acidocaldarius DSM 639
DSM #88
4
(S)-9b/n.d.c
n.d.c
a Time = 5 d. b GC-analysis. c n.d. = not determined due to exceedingly low conversion.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 2 – 2 6 5 6
2 6 5 3

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Fig. 1 Medium optimisation for Sulfolobus acidocaldarius DSM 639 by variation of the carbon source.
Table 2 Enantioselectivities of the microbial hydrolysis of sec-alkyl sulfate esters rac-1a–9a using Sulfolobus acidocaldarius DSM 639 aerobically
grown on sucrose-supplemented Brock-mediuma
Substrate
Conversion (%)
Product/ee (%)
Enantioselectivity (E-value)
rac-1a
rac-2a
rac-3a
rac-4a
rac-5a
32
40
43
32
38
(S)-1b/>99
(S)-2b/59
(S)-3b/55
(S)-4b/62
(S)-5b/65
>200
6
5
6
7
rac-6a
rac-7a
rac-8a
rac-9a
42
10
11
7
(S)-6b/>99
(S)-7b/16
(S)-8b/16
9b/n.d.b
>200
<2
<2
n.d.b
(R)-1ac
40
(S)-1b/ꢀ97
n.a.d
a Time = 5 d. b n.d. = not determined due to low conversion. c ee ꢀ 97%. d n.a. = not applicable.
sulfatase RS2 from Rhodococcus ruber DSM 44541 showed
only E = 21 for rac-1a in the absence of enzyme inhibitors.16
When the sulfate ester moiety was gradually moved towards the
center of the molecule (3-octyl-sulfate, rac-2a; 4-octyl sulfate,
rac-3a), the enantioselectivities for S. acidocaldarius DSM 639
decreased due to the fact that the alkyl groups flanking the
sulfate ester group became similar in size, thus making the chiral
recognition process more difficult. We were particularly pleased
to see that the sterically demanding substrate rac-6a (which was
not accepted by sulfatase RS2 16) was not only well accepted, but
also displayed excellent enantioselectivity.
In order to prove the stereochemical course of the hydrolysis
with respect to retention or inversion of configuration, (R)-2-
octyl sulfate 1a (ee ꢀ 97%) was hydrolysed with optimised
S. acidocaldarius DSM 639 cells. As the sole product, (S)-2-
octanol (ee ꢀ 97%) was detected indicating a highly desired
complete inversion of configuration. The enantio-preference of
S. acidocaldarius DSM 639 for substrates rac-1a–8a revealed a
homogeneous picture, i.e. the (R)-enantiomers were preferen-
tially hydrolysed to furnish the corresponding (S)-configurated
sec-alcohols.
(R)-7b and (R)-8b were synthesized by the following method
(Scheme 3). Starting from amino acids (S)-7c and (S)-8c, various
attempts were undertaken to synthesize (R)-7b and (R)-8b via the
corresponding a-hydroxy acids (S)-7d and (S)-8d in a single-step
reduction based on a protocol of Gevorgyan et al.32 Thus, the
a-hydroxyl group of (S)-7d and (S)-8d was protected as benzyl
ether33 and the carboxylic acid moiety was then subsequently
reduced using HSiEt3 in the presence of a catalytic amount of
B(C6F5)3. The reduction, however, failed. Alternatively, single-
step reduction of the a-hydroxy acid methyl ester,34 or the non-
protected a-hydroxy acid and the a-hydroxy acid methyl ester
were unsuccessful. In a different approach, dehalogenation of
the p-halogen using Pd on carbon failed likewise.35 Finally,
reduction of (S)-7d and (S)-8d by LiAlH4 gave diols (S)-7e and
(S)-8e, which were transformed via the corresponding tosylates
(S)-7f and (S)-8f into (R)-7b and (R)-8b.
Conclusions
In summary, whole cells of hyperthermophilic sulfur-
metabolizers, such as Sulfolobus solfataricus DSM 1617, Sul-
folobus shibatae DSM 5389 and Sulfolobus acidocaldarius DSM
639, were identified as a convenient source of sec-alkylsulfatase
activity with enhanced enantioselectivities and a wider substrate
spectrum compared to Rhodococcus sulfatase RS2, when grown
on a sucrose-supplemented Brock-medium. The full substrate-
selectivity pattern of these novel biocatalysts is currently being
studied.
Determination of absolute configuration
The absolute configuration of products 1b–8b was determined by
co-injection of commercially available or independently synthe-
sized reference samples on GC using a chiral stationary phase.
(S)-4-Octanol was obtained by coupling of ethylmagnesium
bromide to (R)-(+)-1,2-epoxyhexane.31 p-Halophenylpropanols
2 6 5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 2 – 2 6 5 6

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Scheme 3 Synthesis of reference material for (S)-3b, (R)-7b and (R)-8b. Reagents and conditions: a) EtMgBr, Li2CuCl4, THF, −78 ◦C to rt, 6 h; b)
NaNO2, H2SO4, 0 ◦C to rt, 18 h; c) LiAlH4, Et2O, reflux to rt, 17 h; d) p-TsCl, pyridine, 0 ◦C to rt, 24 h; e) LiAlH4, Et2O, 0 ◦C to rt, 3 h.
Determination of conversion
Experimental
An aliquot of 1 mL from the reaction mixture was extracted
with 600 lL of ethyl acetate. After vigorous vortexing (30 s),
centrifugation and drying over Na2SO4, 350 lL of the organic
phase was mixed with 470 lL ethyl acetate and 70 lL of a stock
solution of 2-dodecanol (1 : 10 in EtOAc) as internal standard
and the conversion was determined by GC on column A.
For general analytical and synthetic methods, the synthesis of
substrates (rac-1a–9a) and reference materials [(S)-3b, (S)-7d,
(S)-8d, (S)-7e, (S)-8e, (S)-7f, (S)-8f, (R)-7b, (R)-8b] and their
spectroscopic data see ESI.†
Biocatalytic procedures
Determination of enantiomeric excess of product
Bacterial strains. All strains except Sulfolobus acidocaldarius
IFO 15159, were obtained from the Deutsche Stammsammlung
von Mikroorganismen, Braunschweig, Germany. Sulfolobus
acidocaldarius IFO 15159 was obtained from the Institute of
Fermentation, Osaka, Japan.
For the determination of the enantiomeric excess, the alcohol
formed during biohydrolysis of the sulfate ester was derivatized
(Ac2O, DMAP, EtOAc) to obtain the corresponding acetate
ester in order to achieve enantioseparation on a chiral GC-
column. An aliquot of the enzyme reaction mixture (300 lL) was
extracted with 400 lL of ethyl acetate and dried over Na2SO4.
After addition of 500 lL of acetic anhydride and cat. DMAP,
the capped Eppendorff vial was shaken at rt for 30 min. Finally,
the samples were extracted with 500 lL of water, the organic
phase was dried over Na2SO4 and the samples were analyzed on
column B. For GC-data see the ESI.†
Strain maintainance. Cultures were frozen and stored in 50%
glycerol solution at −80 ◦C.
Anaerobic cultures. Strains were grown according to the
recommended DSMZ media in standing cultures (50 ml flasks
flushed with N2 and capped with a septum) at 80 or 90 ◦C
depending on the optimal temperature conditions in a conven-
tional thermostated drying oven.
Acknowledgements
Aerobic cultures. Sulfolobales strains were grown in Brock’s
basal salts mixture:30 1.3 g (NH4)2SO4, 0.28 g KH2PO4, 0.25 g
MgSO4·7H2O, 0.07 g CaCl2·2H2O, 0.02 g FeCl3·6H2O, 1.8 mg
MnCl2·4H2O, 0.22 mg ZnSO4·7H2O, 0.05 mg CuCl2·2H2O,
0.03 mg VOSO4·2H2O, 0.01 mg CoSO4·6H2O, 0.03 mg
Na2MoO4·2H2O and 4.5 lg Na2B4O7·10H2O, per liter, supple-
mented with 1 g of yeast extract. Additional sugars (glucose,
glycerol, mannitol, sucrose) were added in amounts of 20 g L−1.
The pH was adjusted to 3–3.5 with 50% aqueous H2S◦O4. All
cells were grown aerobically in standing cultures at 70 C in a
conventional thermostated drying oven.
This study was performed in cooperation with Degussa AG
(Frankfurt) within the Research Centre Applied Biocatalysis
and financial support by the FFG, the City of Graz and the
Province of Styria is gratefully acknowledged. G. Antranikian
and his crew are cordially thanked for their excellent advice in
the complex microbiology of Archaea.
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General screening procedure
Rac-2-octyl sulfate (50 g L−1), rac-1-phenyl-2-propyl sulfate
(50 g L−1) and rac-6-methyl-5-hepten-2-yl sulfate (50 g L−1) were
selected as test substrates for a screening for new alkylsulfatase
activity at different temperatures: rt (anaerobic); rt, 60, 85 and
95 ◦C, respectively (aerobic). 200 lL of the sulfate ester solution
(10 mg sulfate, 200 ll Tris buffer, pH 7.5, 50 mM) was added to
1 mL of the culture and the mixture was shaken at 140 rpm at
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27 Anaerobically-grown strains: Desulfurococcus amylolyticus DSM
3822, Pyrobaculum organotrophum DSM 4185, Pyrococcus woesei
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