The substrate spectrum of the inverting sec-alkylsulfatase Pisa1

Article abstract of DOI:10.1002/adsc.201100864

The substrate spectrum of the inverting alkylsulfatase Pisa1 was investigated using a range of sec-alkyl sulfate esters bearing aromatic, olefinic and acetylenic moieties. Perfect enantioselectivities were obtained for substrates bearing groups of different size adjacent to the sulfate ester moiety. Insufficient selectivities could be doubled by using dimethyl sulfoxide (DMSO) as co-solvent. Hydrolytically unstable benzylic sulfate esters could be sufficiently stabilised by introduction of electron-withdrawing substituents. Overall, Pisa1 appears to be a very useful inverting alkylsulfatase for the deracemisation of rac-sec-alcohols via enzymatic hydrolysis of their corresponding sulfate esters, which furnishes homochiral products possessing the 'anti-Kazlauskas' configuration. Copyright

Full text of DOI:10.1002/adsc.201100864

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DOI: 10.1002/adsc.201100864
The Substrate Spectrum of the Inverting sec-Alkylsulfatase Pisa1
Markus Schober,a Tanja Knaus,b Michael Toesch,a Peter Macheroux,b
Ulrike Wagner,c and Kurt Fabera,*
a
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Fax : (+43)-316-380-9840; phone: (+43)-316-380-5332; e-mail: Kurt.Faber@Uni-Graz.at
Institute of Biochemistry, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria
Institute of Molecular Biosciences, Structural Biology, University of Graz, Humboldtstrasse 50/III, A-8010 Graz, Austria
b
c
Received: November 4, 2011; Revised: February 15, 2012; Published online: June 5, 2012
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201100864.
Abstract: The substrate spectrum of the inverting al- by introduction of electron-withdrawing substituents.
kylsulfatase Pisa1 was investigated using a range of Overall, Pisa1 appears to be a very useful inverting
sec-alkyl sulfate esters bearing aromatic, olefinic and alkylsulfatase for the deracemisation of rac-sec-alco-
acetylenic moieties. Perfect enantioselectivities were hols via enzymatic hydrolysis of their corresponding
obtained for substrates bearing groups of different sulfate esters, which furnishes homochiral products
size adjacent to the sulfate ester moiety. Insufficient possessing the ꢀanti-Kazlauskasꢁ configuration.
selectivities could be doubled by using dimethyl sulf-
oxide (DMSO) as co-solvent. Hydrolytically unstable Keywords: alkylsulfatase; deracemization; inversion;
benzylic sulfate esters could be sufficiently stabilised substrate spectrum
Introduction
(alkyl) sulfatases.[10] It was only recently that they
have been identified as members of the metallo-b-lac-
Driven by the demand to enhance the economic bal- tamase family, whicÀh act through nucleophilic dis-
ance of chemical processes, the design of asymmetric placement of HSO4 via (formal) [OHÀ], which is
catalytic protocols which allow the transformation of provided from water by a binuclear Zn2+-cluster.[11]
a racemate into a single stereoisomeric product with- We have recently characterised the first inverting sec-
out the occurrence of an ꢀunwantedꢁ isomer is an im- alkylsulfatase (Pisa1) from Pseudomonas sp. DSM
portant topic.[1] Depending on the type of starting ma- 6611 on a molecular level.[12,13] In order to evaluate
terial, these so-called deracemisation methods are the applicability of this useful enzyme, which trans-
based on dynamic kinetic resolution,[2] stereo-inver- forms a rac-sec-alkyl sulfate into a homochiral mix-
sion[3] or enantio-convergent processes.[4] In the latter, ture of a sec-alcohol and non-reacted sulfate ester,
both starting enantiomers are transformed via inde- the substrate tolerance and the operational stability
pendent pathways via retention or inversion of config- of this enzyme are reported here.
uration to furnish a single stereoisomer. In order to
fulfil this prerequisite, catalysts must not only be
enantioselective (by preferring one enantiomer over Results and Discussion
the other), but also stereoselective (by inverting one
enantiomer, but retaining the other). Enzymes that In order to provide a broad coverage of substrate
elicit the complex potential to invert the stereochem- structures, sec-alkyl sulfates of varying chain length
istry of their substrate during catalysis are rather rare (rac-1a–8a) were chosen with special emphasis on the
and encompass haloalkane dehalogenases,[5] epoxide relative position of the sulfate ester moiety ranging
hydrolases[6, and (alkyl) sulfatases.[7] Depending on from (w-1) to (w-3) (Scheme 1). The latter substrates
the subtype of enzyme, enzymatic sulfate ester hy- bearing an ꢀinternalꢁ (w-3)-functionality are difficult
drolysis may proceed via cleavage of the S O or the to tackle with traditional lipase[14] or carbonyl reduc-
À
C O bond, which causes retention or inversion of tase technology.[15] From a synthetic viewpoint, sub-
À
configuration at the stereogenic carbon centre.[8] strates bearing lipophilic alkene or alkyne functionali-
Whereas the mechanism of retaining sulfatases is well ties (rac-9a–20a) allow access to valuable building
understood,[9] much less is known about inverting blocks via functional group manipulation via (stereo)-
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Scheme 1. Kinetic resolution of sec-alkyl sulfate esters rac-1a–24a using the inverting alkylsulfatase Pisa1 yields homochiral
products.
selective redox-reactions. Finally, two stereochemical- according to literature methods. The absolute configu-
ly sensitive benzylic sulfate esters were selected (rac- rations of compounds 3b, 14b and 15b were deter-
23a, rac-24a). Since Pisa1 acts via a single pathway mined via optical rotation measurements. Alcohols
through strict inversion of configuration, as proven by 16b, 18b and 19b were correlated via catalytic hydro-
18O-labelling experiments,[12] E values can be applied genation yielding 2b, 3-hexanol and 7b, respectively.
for the description of the enantioselectivity.[16] Since Compounds 4b, 6b, 8b and 22b were elucidated as re-
the formed product (a lipophilic alcohol) and the cently reported.[12]
non-reacted (water-soluble) sulfate ester enantiomers
The substrate-selectivity pattern revealed several
have extremely different characteristics with respect clear trends (Table 1). Overall, substrates bearing the
to their relative solubilities, E values were calculated sulfate ester group in the (w-1)-position show that the
from the enantiomeric excess of substrate and product enantioselectivity strongly depends on the relative
(eeS, eeP), because the latter are relative values and size of both alkyl substituents: With increasing chain
are thus (almost) independent of sample manipula- length or branching, E values steadily climbed from
tions.[17,18] The overall procedure was as follows 1a [E=16 via 2a (E=155) to 3a–5a (E>200)]. Anal-
(Scheme 1). In a first step, racemic substrates were ogous observations were made when the functional
subjected to enzymatic hydrolysis in Tris-buffer at the group was gradually moved towards the centre of the
pH optimum of 8.2. After the conversion came close molecule: Whereas the (w-2)-substrate 6a was nicely
to mid-point (depending on the reactivity of the sub- resolved, compounds 7a and 8a showed reduced ste-
strate 2–72 h), the formed alcohol was separated from reoselectivities due to the decreasing size difference
the non-reacted sulfate ester by extraction. The latter of both alkyl substituents. Similar trends can be de-
was hydrolysed under acidic conditions, which pro- duced from substrates bearing a terminal olefinic
ceeds with strict retention of configuration.[19] Dera- group (9a, 10a).
cemisation experiments were performed by linking
When the position of the alkene unit was switched
both hydrolysis steps in a sequential fashion (without to yield allylic sulfate esters 11a–14a, E values were
separation of intermediates) in a one-pot procedure surprisingly modest (E=17 to 149). In order to en-
using substrate rac-4a.
hance the stereoselectivity of the reaction by sup-
The absolute configuration of products was eluci- pressing the background of slow (non-enzymatic) au-
dated by co-injection of reference samples of alcohols tohydrolysis of the substrate,[25] a range of organic co-
1b, 2b, 5b, 7b,[20] 9b, 10b, 11b,[21] 12b, 13b,[21] 17b,[22] solvents was tested using rac-12a as test substrate
20b,[23] 21b,[24] 23b and 24b, which were obtained com- (Figure 1). Whereas water-immiscible co-solvents,
mercially or were prepared via independent syntheses such as toluene, and cyclohexane had little effect on
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The Substrate Spectrum of the Inverting sec-Alkylsulfatase Pisa1
Table 1. Kinetic resolution of alkyl sulfates rac-1a–24a using the inverting sec-alkylsulfatase Pisa1.
Substrate
Time [h]
Cosolvent
Conversion [%][a]
eeP
eeS
Configuration[b]
E Value[a]
rac-1a
rac-2a
24
24
24
2
24
6
none
none
none
none
none
none
none
none
none
none
none
DMSO
none
DMSO
none
DMSO
none
DMSO
none
none
none
DMSO
none
none
none
none
none
none
DMSO
none
34
39
50
50
50
50
34
57
7
50
35
45
48
47
50
49
50
46
44
4
49
47
57
56
23
24
50
n.d
n.d
10
13
83
98
42
62
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
16
155
rac-3a
>99
>99
>99
>99
96
60
94
>99
83
88
90
99
94
99
95
99
74
>99
93
98
55
32
91
>99
>99
33
81
>99
>99
>99
>99
>99
>99
49
80
7
>99
46
74
87
87
94
95
96
83
58
4
89
89
80
40
27
31
>99
n.d
n.d.
11
>200
>200
>200
>200
87
10
34
>200
17
36
102
>200
125
>200
149
>200
12
>200
80
>200
8
3
28
rac-4c[c]
rac-5a
rac-6a[c]
rac-7a
6
rac-8a[c]
rac-9a
24
24
24
6
8
6
8
6
8
6
(S)
(S)
rac-10a
rac-11a
rac-11a
rac-12a
rac-12a
rac-13a
rac-13a
rac-14a
rac-14a
rac-15a
rac-16a
rac-17a
rac-17a
rac-18a
rac-19a
rac-20a
rac-21a
rac-22a[c]
rac-23a[e]
rac-23a[e]
rac-24a
rac-24a
(R)[d]
(R)[d]
(R)[d]
(R)[d]
(R)[d]
(R)[d]
(R)[d]
(R)[d]
(S)
8
24
72
6
6
4
(S)
(S)
(S)
(S)
6
(S)
24
24
24
6
6
72
72
(R)[d]
(R)[d]
(R)[d]
(S)
>200
>200
n.d
n.d
>200
>200
(S)
(S)
(S)
DMSO
14
[a]
Calculated from eeS and eeP.[18]
[b]
Since enzymatic hydrolysis of the preferred (R)-enantiomer proceeds with strict inversion of configuration, the alcohol
formed and the non-reacted sulfate ester possess the same absolute configuration.
Data taken from ref.[12]
Switch in CIP priority.
Not determined due to competing (non-enzymatic) autohydrolysis.
[c]
[d]
[e]
the catalytic performance of Pisa1, t-BuOMe, ethyl
With (w-1)-substrates bearing an internal alkyne
acetate and chlorinated hydrocarbons caused a drop unit (15a–17a) the relative position of the acetylene
in activity and selectivity. In contrast, some water-mis- moiety had a strong impact on the selectivity (15a,
cible solvents, such as ethylene glycol, DMSO, 1,2-di- 16a) as well as the size-difference of substituents (18a,
methoxyethane and methanol led to an increase of se- 19a). The selectivity of 17a could be drastically in-
lectivity (eeP from 90% to >99%) at a small cost of creased from E=80 to >200. In contrast to more
relative activity (~10%). Other solvents caused a dra- flexible allylic substrates, rigid propargylic sulfate
matic loss of activity. Based on these data, DMSO esters (21a, 22a) were resolved with excellent selectiv-
(20% v:v) turned out to be the best selectivity en- ity, as long as both substituents differed in size. Un-
hancer by roughly doubling E values of substrates fortunately, the benzylic sulfate 23a proved to be hy-
11a–14a. The exact molecular reason for the selectivi- drolytically unstable and showed spontaneous (non-
ty-enhancing effects exerted by DMSO can be attrib- enzymatic) hydrolysis, which was assumed to proceed
uted to suppression of spontaneous (non-enzymatic) via an SN1 mechanism involving a benzylic carbenium
hydrolysis and/or alteration of the catalytic properties ion derived by departure of the good leaving group
À
of the enzyme, as observed for an alkyl sulfatase from HSO4 . In order to prove this hypothesis, electron-
Rhodococcus ruber DSM 44541.[26]
withdrawing CF3 substituents (which impede reso-
nance stabilisation) were added. Indeed, substrate 24a
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Figure 1. Relative conversion and enantioselectivity of Pisa1 for rac-12a in the presence of organic cosolvents. Reaction con-
ditions: rac-12a (5 mg, 22 mM), Pisa1 (20 mL, 130 mg, 1.8 mM), Tris-HCl buffer (100 mM, pH 8.0, 20% v/v organic cosolvent),
reaction volume: 1000 mL, 308C, 120 rpm, 6 h.
turned out to be perfectly stable and thus could be re- of sec-alkyl sulfate esters were accepted. Aromatic,
solved with excellent enantioselectivity.
olefinic and acetylenic moieties, which allow further
Temperature stability tests using substrate rac-4a functional group manipulation, were nicely tolerated.
with pre-incubated Pisa1 revealed that the enzyme Perfect enantioselectivities were obtained for sub-
was very stable at 20–308C (24 h incubation), which strates bearing groups of different size adjacent to the
caused only a small loss of activity (ꢀ10%). However, sulfate ester moiety. For stereochemically ꢀdifficultꢁ
incubation at 408C caused an activity loss of ~20% substrates possessing substituents of similar size, in-
and at 508C significant enzyme deactivation was ob- sufficient selectivities could be significantly enhanced
served. The latter was underlined by detailed kinetic by using DMSO as co-solvent. Hydrolytically labile
studies performed at 20, 30 and 408C, which revealed benzylic sulfate esters, which are impeded by back-
a deterioration of catalytic parameters (rising KM, de- ground hydrolysis, could be sufficiently stabilised by
creasing kcat) at elevated temperatures. A melting introduction of electron-withdrawing groups. Overall,
point of 578C was determined for Pisa1 by CD meas- Pisa1 proved to be a very useful inverting alkylsulfa-
urements.
tase for the deracemisation of rac-sec-alcohols via en-
zymatic hydrolysis of their corresponding sulfate
esters, which furnishes homochiral products. Although
Pisa1 exhibits the same stereochemical preference for
sec-alkyl sulfate esters as lipases for sec-alcohol car-
Conclusions
Investigation of the substrate spectrum of the invert- boxylic esters (cf. Kazlauskas rule[27]), anti-Kazlauskas
ing alkylsulfatase Pisa1 revealed that a wide variety products, which are accessible by using subtilisin-
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Adv. Synth. Catal. 2012, 354, 1737 – 1742

The Substrate Spectrum of the Inverting sec-Alkylsulfatase Pisa1
based dynamic kinetic resolution,[28] are formed via
inversion of configuration.
were added. The mixture was stirred under reflux at 408C
for 2 h. After cooling to room temperature, saturated
NaHCO3 solution (5 mL) was added to quench the reaction.
After phase separation the organic phase was dried over an-
hydrous sodium sulfate. The solvent was evaporated under
reduced pressure, the remaining alcohol was redissolved in
ethyl acetate (1 mL), derivatised as acetate[12] and analysed
by GC using a chiral stationary phase.
Experimental Section
General Remarks
Competent cells One Shotꢃ TOP10 and One Shotꢃ BL21
Starꢄ (DE3) were obtained from Invitrogen and trans-
formed according to the protocol of the supplier. Alcohols
rac-1b–24b, the respective enantiopure alcohol references
and all other reagents were purchased from Sigma Aldrich,
Acros, Carl Roth and Alfa Aesar. NMR spectra were re-
corded on a Bruker spectrometer at 300 (1H) and 75 (13C)
MHz. Shifts (d) are given in ppm and coupling constants (J)
are given in Hz.
Enantioconvergent One-Pot Two-Step Hydrolysis of
rac-2-Octyl Sulfate (4a)
rac-2-Octyl sulfate (4a, 10 mg) was hydrolysed using Pisa1
(0.19 mg) in Tris/HCl buffer (0.1M, pH 8.0, 2 mL) by shak-
ing at 308C and 120 rpm for 6 h in a closed glass vial.
Methyl tert-butyl ether (2 mL), p-TosOH (60 mg) and 1-oc-
tanol (2 mg, as internal standard) were added and the mix-
ture was incubated for 24 h with shaking at 408C or 608C,
respctively. After cooling to 08C, saturated NaHCO3 solu-
tion was added (1 mL) to neutralise the acid, the mixture
was centrifuged, the organic layer was separated, dried
(Na2SO4) and derivatised as acetate as described above. GC
analysis showed that the relative accuracy of the conversion
from double experiments was Æ7% using a calibration
curve. Alcohol (S)-4b was obtained as the sole product
(average values from double experiments): (i) T=408C, c=
78%, ee 97.5%; (ii) T=608C, c=93%, ee 98.7%.
Synthesis of Alkyl Sulfate Esters
Racemic sulfate esters 1a–3a, 5a, 7a, 9a–21a, 23a and 24a
were prepared from the corresponding alcohols 1b–3b, 5b,
7b, 9b–21b, 23b and 24b by using NEt3·SO3 following
a known procedure[12] with the following modification: NaH
(60% suspension in paraffin oil) was added directly in equi-
molar amounts.
The use of microwave heating for the acid-catalysed hy-
drolysis step could not be employed due to significant race-
misation: (R)-2-octyl sulfate (4a) in Tris/HCl buffer (0.1M,
pH 8.0, 1 mL) containing p-TosOH (30 mg) was placed in
a sealed vial and heated for 15 min to 1008C, 1108C, 1208C
and 1808C, respectively. After cooling to 08C, EtOAc
(1 mL) containing 1-octanol (1 mg, as internal standard) was
added. The organic layer was separated, dried (Na2SO4) and
derivatised as acetate as described above. GC analysis re-
vealed that the enantiomeric purity of (R)-4b was signifi-
cantly diminished. 1008C: ee 44%; 1108C: ee 36%; 1208C:
ee 39%; 1808C: ee 49%, respectively.
Cloning, Expression and Purification of Alkyl-
sulfatase Pisa1 from Pseudomonas sp. DSM6611
Pisa1 was obtained according to a known procedure.[12]
Screening for Activity and Determination of
Stereoselectivity
The activity of Pisa1 was tested using alkyl sulfate esters
rac-1a–3a, rac-5a, rac-7a, rac-9a–21a, rac-23a and rac-24a
(5 mg) as previously described.[12] Depending on the relative
activity, reaction times of 2–72 h were applied.
For the calculation of the conversion and E values from
eeP and eeS (after acid-catalysed hydrolysis of the non-react-
ed sulfate ester with retention of configuration yielding the
corresponding alcohol) a previously described two-step pro-
tocol[12] was employed with the following modifications:
Step 1 (enzymatic hydrolysis): Alkyl sulfate (1a–24a,
25 mg) was dissolved in Tris-HCl (4.9 mL, 100 mM, pH 8.2)
and a solution of purified enzyme (0.65 mg, 8.85 nmol pro-
tein in 0.1 mL) was added. The reaction mixture was shaken
at 308C and 120 rpm for 2–72 h. After extraction with ethyl
acetate (2 mL), an aliquot of the organic layer (1 mL) was
Supporting Information
NMR data and yields of substrates, determination of enan-
tiomeric excess and absolute configuration of products, GC
analytics, optical rotation values, detailed procedures for co-
solvent, temperature stability and pH studies as well as de-
termination of protein concentration are given in the Sup-
porting Information.
dried over anhydrous sodium sulfate, derivatised to the cor- Acknowledgements
responding acetate 1c–24c, which was analysed by GC using
a chiral stationary phase. The aqueous phase was washed
twice with ethyl acetate (3 mL) to remove traces of alcohol
and was lyophilised overnight. When DMSO was used as
a co-solvent, the extraction was repeated 3 times with ethyl
acetate (5 mL) to remove residual traces of DMSO before
lyophilisation.
This study was financed by the Austrian Science Fund within
the DK Molecular Enzymology (FWF, project W9). The au-
thors would like to thank Franz Mlynek, Cathrin Zeppek,
Nina Schmidt, Kerstin Prettler and Petra Gadler for their val-
uable assistance and Oliver Kappe and Bartholomꢀus Pieber
for advice in microwave technology.
Step 2 (acidic hydrolysis):[19] The lyophilisate was dis-
solved in a mixture of methyl tert-butyl ether/deionised
water (10 mL, 97:3) and 1,4-dioxane (5 mL, 0.06 mmol) and
p-toluenesulfonic acid monohydrate (170 mg, 0.9 mmol)
Adv. Synth. Catal. 2012, 354, 1737 – 1742
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Adv. Synth. Catal. 2012, 354, 1737 – 1742