Hydrolysis of Benzylic sec-Sulfate Esters
dried with Na2SO4 and alcohols 3b–6b and 8b were derivatized to
the corresponding acetates with DMAP (1 mg) and acetic anhy-
dride (100 μL) overnight. The reaction was quenched by addition
of H2O (300 μL) with stirring for 3 h. After centrifugation for 3 min
at 13.000 rpm, the organic phase was dried with Na2SO4 and di-
rectly measured with GC-FID. The enzymatic hydrolysis of sub-
strates 1a, 2a and 7a is described elsewhere.[8]
data confirm that Pisa1 hydrolyzed (R)-5a with complete
inversion with concomitant incorporation of 18O at C
within the limits of accuracy (calculated 84:16, measured
79:21).
The pathways of autohydrolysis were investigated by an
analogous experiment in the absence of enzyme by using an
18/16O-label of 83:17 at a fivefold-extended reaction time.
The following facts were deduced:
Quantification of Autohydrolysis: The respective sulfate ester 3a–6a
and 8a was dissolved in Tris/HCl-buffer (1 mL, 100 mm, pH 8.0)
and were shaken at 120 °C and 30 rpm for 24 h. The reaction was
quenched by freezing in liquid N2 and was thawed individually
prior to measurement. Quantification of autohydrolysis was done
from calibration curves with the corresponding alcohol and sulfate
ester.
(i) Non-enzymatic hydrolysis of enantiopure (R)-5a (e.r.
Ͼ99/Ͻ1) gave (S)-5b with an e.r. of 81:19, indicating that
inversion through SN2 at C is a dominant pathway.
(ii) The (R)-enantiomer of alcohol 5b derived from (R)-5a
can either be formed through retention or racemization, but
18O-labeling of (R)-5b can only take place through racemi-
zation, because retention retains the 16O-label. Because the
ratio of 18/16O-label in (R)-5b (79:21) corresponds to that of
the aqueous medium (83:17) within the accuracy of mea-
surement, it can be concluded that retention at C through
SN2 at S can be neglected and racemization through SN1
through a benzylic carbenium ion strongly prevails.
All measurements were carried out with a Shimadzu HPLC system
(CBM-20A, LC-20AD, DGU-20A5, SIL-20AC, CTO-20AC, SPD-
M20A, CBM-20A) by using a ZORBAX 300-SCX (4.6ϫ250 mm)
IEX column and UV-detection [diode array detector set at 271 nm
(3a), 261 nm (4a), 266 nm (5a), 262 nm (6a) and 259 nm (8a)]. The
conversion was determined by using sodium formate buffer
(200 mm pH 2.8) at a flow rate of 0.5 mL/min and a run time of
20 min (for retention times see Supporting Information, Table S1).
18O-Labeling Experiments: 18O-Enriched water (90 μL, 18O content
97%) was added to a buffer solution (16OH2 10 μL, 1 m Tris/HCl
pH 8.0) to reach a final buffer concentration of 100 mm (18/16O-
label 83:17). Substrate (R)-5a (1 mg) was added to the solution and
was shaken for 24 h at 30 °C and 120 rpm. Afterwards, alcohol 5b
was extracted with ethyl acetate (0.1 mL), the organic phase was
dried with Na2SO4 and directly measured with GC–MS. GC–MS
measurements were carried out with an Agilent 5975C MS con-
nected to an Agilent 7890A GC fitted with a CTC Analytics
(iii) Consequently, inversion (SN2 at C) and racemization
(SN1) are the major pathways. Their relative proportion can
be estimated by taking the erosion of e.r. from (R)-5a to (S)-
5b (e.r. from Ͼ99R:Ͻ1S to 81S:19R) into account: Because
racemization produces equal amounts of (R)- and (S)-5b
(19 parts each, i.e. 38 in total), the remainder of 62 parts
counts for inversion (considering retention below the limits
of detectability Յ 3). Consequently, the ratio of relative
rates of kinv (SN2 at C) versus krac (SN1) are about 1.6:1.
PAL Autosampler by using
a Chirasil Dex CB column
(25 mϫ0.32 mmϫ0.25 μm film) and He as a carrier gas (0.69 bar).
Injection temperature 250 °C, flow 0.5 mL/min, temperature pro-
gram: 80° hold 1 min, 15 °C/min to 141 °C, 0.5 °C/min to 143 °C,
17 °C/min to 180 °C. Retention times: (R)-5b 7.4 min, (S)-5b
7.7 min.
Conclusions
The enantioselectivity of the enzymatic hydrolysis of
benzylic sec-sulfate esters by using inverting alkylsulfatase
Pisa1 could be significantly improved by suppressing the
autohydrolysis of substrates by addition of DMSO as co-
solvent. H218O-Labeling studies revealed that the major
pathway of autohydrolysis proceeded through SN2-type in-
version at carbon. In contrast, nucleophilic attack at sulfur
and the SN1-type pathway through a benzylic carbenium
ion took place at the limits of detection. The data obtained
are interpreted by analysis of Hammett constants of meta
substituents. These results contribute to the understanding
of the bioactivity of sulfated steroids possessing carcino-
genic[23] or anabolic properties[24] and the stereo-comple-
mentary nucleophilic substitution of sulfur-based leaving
groups.[25]
Enzymatic reactions were performed analogously to the control re-
action with addition of Pisa1 (26 μg, 353 pmol, 4.6 μL of stock
solution in 16OH2).
Supporting Information (see footnote on the first page of this arti-
cle): Expression of PISA1, synthesis of substrates and reference
compounds, analytical methods, NMR and MS spectra, and op-
tical rotation values are presented.
Acknowledgments
This project was performed within DK Molecular Enzymology and
financial support by the Austrian Science Fund (FWF) (project
W9) is gratefully acknowledged
[1] U. T. Bornscheuer, R. J. Kazlauskas, Hydrolases in Organic
Synthesis: Regio- and Stereoselective Biotransformations, 2nd
ed., Wiley-VCH, Weinheim, Germany, 2006.
[2] B. Martin-Matute, J.-E. Bäckvall, Curr. Opin. Chem. Biol. 2007,
11, 226–232; J. H. Lee, K. Han, M.-J. Kim, J. Park, Eur. J. Org.
Chem. 2010, 999–1015.
[3] N. J. Turner, Curr. Opin. Chem. Biol. 2010, 14, 115–121; K.
Faber, Chem. Eur. J. 2001, 7, 5004–5010.
[4] M. Schober, K. Faber, Trends Biotechnol. 2013, 31, 468–478.
[5] M. Schober, M. Toesch, T. Knaus, G. A. Strohmeier, B.
van Loo, M. Fuchs, F. Hollfelder, P. Macheroux, K. Faber, An-
Experimental Section
Enzymatic Hydrolysis of Sulfate Esters 3a–6a and 8a: The corre-
sponding sulfate ester 3a–6a and 8a (5 mg) was dissolved in Tris/
HCl buffer (1 mL, 100 mm, pH 8.0), Pisa1 was added (0.13 mg) and
the reaction was shaken with 120 rpm for 24 h at 30 °C. Afterwards,
ethyl acetate (1 mL) was added and the mixture was centrifuged
for 3 min at 13.000 rpm. The organic phase was separated and
Eur. J. Org. Chem. 2014, 3930–3934
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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