Highly enantioselective sec-alkyl sulfatase activity of Sulfolobus acidocaldarius DSM 639

Article abstract of DOI:10.1021/ol0477778

(Chemical Equation Presented) rac-sec-Alkyl sulfate esters 1a-4a were resolved in high enantioselectivities with E-values up to >200 using whole cells of aerobically grown Sulfolobus acidocaldarius DSM 639. 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.

Full text of DOI:10.1021/ol0477778

ORGANIC
LETTERS
2004
Vol. 6, No. 26
5009-5010
Highly Enantioselective sec-Alkyl
Sulfatase Activity of Sulfolobus
acidocaldarius DSM 639
Sabine R. Wallner, Bettina M. Nestl, and Kurt Faber*
Department of Chemistry, Organic and Bioorganic Chemistry, UniVersity of Graz,
Heinrichstrasse 28, A-8010 Graz, Austria
kurt.faber@uni-graz.at
Received October 29, 2004
ABSTRACT
rac-sec-Alkyl sulfate esters 1a−4a were resolved in high enantioselectivities with E-values up to >200 using whole cells of aerobically grown
Sulfolobus acidocaldarius DSM 639. 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.
Sulfatases catalyze the hydrolytic cleavage of the sulfate ester
bond.1 In contrast to the majority of hydrolytic biotransfor-
mations catalyzed by lipases, esterases, and proteases, which
do not alter the stereochemistry of the substrate, the
stereochemical course of sulfate ester hydrolysis can be
controlled by the choice of the appropriate subtype of
sulfatase enzyme: Thus, whereas aryl sulfatases generally
act through retention of configuration at the sulfated carbon
atom by cleavage of the S-O bond,2 alkyl sulfatases lead
to inVersion of configuration by acting on the C-O bond.3,4
This rarely observed phenomenon makes them particularly
attractive for the design of so-called deracemization pro-
cesses, which allow the transformation of a racemate into a
single stereochemical product in 100% theoretical yield.5 This
can be accomplished by removal of the sulfate ester moiety
from the remaining nonconverted sulfate ester by acid-
catalyzed hydrolysis with retention of configuration.6
On the basis of vague hints on the stereospecific and
enantioselective hydrolysis of alkyl sulfate esters,3 we
recently reported an alkyl sulfatase (termed “RS2”) from
Rhodococcus ruber DSM 44541.7,8 On one hand, the enzyme
displayed absolute stereospecificity by acting with strict
inversion of configuration of simple sec-alkyl esters, but its
enantioselectiVity was less than perfect: although 2-octyl
sulfate (rac-1a) was resolved with an acceptable E-value of
21, no appreciable enantioselectivities were observed for 3-
and 4-octyl sulfate (E < 5).7 Attempts to enhance selectivities
by enzyme inhibition (e.g., addition of Fe3+)9 were successful
but (as usual in this technique) led to a significant loss of
catalytic activities. Furthermore, the substrate tolerance of
sulfatase RS2 was rather narrow, as substrates bearing bulky
aryl groups (e.g., rac-4a) were not accepted.7
* Corresponding author. Phone: +43-316-380-5332. Fax: +43-316-380-
9840.
Our search for novel (and more selective) alkyl sulfatases
was led by the idea that organisms known to possess a rich
(1) Dodgson, K. S.; White, G. F.; Fitzgerald, J. W. Sulfatases of Microbial
Origin; CRC Press: Boca Raton, FL, 1982; Vols. 1 and 2.
(2) Boltes, I.; Czapinska, H.; Kahnert, A.; von Bu¨low, R.; Dierks, T.;
Schmidt, B.; von Figura, K.; Kertesz, M. A.; Uson, I. Structure 2001, 9,
483-491.
(6) Wallner, S. R.; Nestl, B.; Faber, K. Tetrahedron 2004, in press.
(7) Pogorevc, M.; Faber, K. Tetrahedron: Asymmetry 2002, 13, 1435-
1441.
(8) Pogorevc, M.; Faber, K. Appl. EnViron. Microbiol. 2003, 69, 2810-
2815.
(9) Pogorevc, M.; Strauss, U. T.; Riermeier, T.; Faber, K. Tetrahedron:
Asymmetry 2002, 13, 1443-1447.
(3) Shaw, D. J.; Dodgson, K. S.; White, G. F. Biochem. J. 1980, 187,
181-196.
(4) Pogorevc, M.; Kroutil, W.; Wallner, S. R.; Faber, K. Angew. Chem.,
Int. Ed. Engl. 2002, 41, 4052-4054.
(5) Faber, K. Chem. Eur. J. 2001, 7, 5004-5010.
10.1021/ol0477778 CCC: $27.50
© 2004 American Chemical Society
Published on Web 12/02/2004

Table 1. Enantioselectivitites of Enzymatic Sulfate Ester Hydrolysis
enantiomeric ratio (E)
substrate
conversion [%]a
product/ee [%]a
Sulfolobus acidocaldarius DSM 639
Rhodococcus sulfatase RS2b
rac-1a
rac-2a
rac-3a
rac-4a
32
40
43
42
(S)-1b/>99
(S)-2b/59
(S)-3b/55
(S)-4b/>99
>200
6
21
4
5
∼1
>200
no conversion
a Data for Sulfolobus acidocaldarius DSM 639. b Data from ref 7.
inorganic sulfur metabolism (encompassing sulfur species
of oxidation states from sulfide to sulfate)10 might also show
sulfated organic species such as alkyl sulfates. From a
screening among anaerobic and aerobic Archaea,11 we
identified whole cells of Sulfolobus acidocaldarius DSM 639
as a convenient source of highly selective alkyl sulfatase
activity. When alkyl sulfate esters rac-1a-4a were subjected
to aerobically grown Sulfolobus cells at pH 2-3, which
corresponds to that of their natural habitats, hydrolysis of
(R)-enantiomers from the racemate provided the correspond-
ing (S)-configured sec-alcohols; in other words, a homochiral
product mixture was generated from the racemate (Scheme
1, Table 1).12 In each case, the absence of undesired
octyl sulfate (ee >97%), which yielded (S)-2-octanol as the
sole product in >97% ee. Excellent enantioselectivity was
observed for 2-octyl sulfate (rac-1a, E > 200); in compari-
son, native sulfatase RS2 showed only E ) 21 in the absence
of inhibitors. When the sulfate ester moiety was gradually
moved toward the center of the molecule (3-octyl- and
4-octyl sulfate, rac-2a, rac-3a), the enantioselectivities
decreased, which is 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-4a (which was not accepted by sulfatase RS2)
was not only well accepted but also displayed excellent
enantioselectivity.
Overall, the enantiopreference of Sulfolobus acidocaldarius
DSM 639 proved to be identical to that of sulfatase RS2,
i.e., (R)-sec-alkyl sulfate esters were hydrolyzed with inver-
sion of configuration to form the corresponding (S)-sec-
alcohols, while (S)-sulfate ester enantiomers remained un-
touched.
Scheme 1. Enantioselective Microbial Hydrolysis of sec-Alkyl
Sulfate Esters with Inversion of Configuration
In summary, whole cells of Sulfolobus acidocaldarius
DSM 639 were identified as a convenient source of alkyl
sulfatase activity with enhanced enantioselectivites and a
wider substrate spectrum as compared to Rhodococcus
sulfatase RS2. The full substrate-selectivity pattern of this
novel biocatalyst is currently being studied.
Acknowledgment. This study was performed in coopera-
tion with Degussa AG (Frankfurt) within the Competence
Center Applied Biocatalysis and financial support by the TIG,
the City of Graz, and the Province of Styria is gratefully
acknowledged. G. Antranikian and his crew is cordially
thanked for their excellent expertise in the microbiology of
Archaea.
spontaneous hydrolysis was verified in the absence of
biocatalyst. The stereochemical course (i.e., clear inversion
of configuration) was proven by the hydrolysis of (R)-2-
Supporting Information Available: Experimental bio-
catalytic procedures and growth conditions. This material is
available free of charge via the Internet at http://pubs.acs.org.
(10) Huber, R.; Huber, H.; Stetter, K. O. FEMS Microbiol. ReV. 2000,
24, 615-623.
(11) Details will be published in a full paper.
(12) For the synthesis of substrates and determination of the absolute
configuration of products see ref 7.
OL0477778
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Org. Lett., Vol. 6, No. 26, 2004