Stereochemistry and mechanism of enzymatic and non-enzymatic hydrolysis of benzylic sec-sulfate esters

Article abstract of DOI:10.1002/ejoc.201402211

The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in ¹⁸O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting S_N2-type nucleophilic attack of [OH^-] at the benzylic carbon represents the major pathway, whereas (ii) S_N1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining S_N2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents. The enzymatic hydrolysis of benzylic sec-sulfate esters by alkylsulfatase Pisa1 proceeded with clean inversion of configuration and with excellent stereoselectivities when the competing non-enzymatic hydrolysis was suppressed by addition of dimethyl sulfoxide as co-solvent. Copyright

Full text of DOI:10.1002/ejoc.201402211

FULL PAPER
DOI: 10.1002/ejoc.201402211
Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis
of Benzylic sec-Sulfate Esters
Michael Toesch,[a] Markus Schober,[a] Rolf Breinbauer,[b] and Kurt Faber*[a]
Keywords: Synthetic methods / Enzyme catalysis / Hydrolysis / Reaction mechanisms / Configuration determination /
Hammett constant
The substrate scope of inverting alkylsulfatase Pisa1 was ex-
tended towards benzylic sec-sulfate esters by suppression of
competing non-enzymatic autohydrolysis by addition of di-
methyl sulfoxide as co-solvent. Detailed investigation of the
mechanism of autohydrolysis in 18O-labeled buffer by using
SN2-type nucleophilic attack of [OH–] at the benzylic carbon
represents the major pathway, whereas (ii) SN1-type forma-
tion of a planar benzylic carbenium ion leading to racemiza-
tion was a minor event, and (iii) Retaining SN2-type nucleo-
philic attack at sulfur took place at the limits of detection.
an enantiopure sec-benzylic sulfate ester as substrate re- The data obtained are interpreted by analysis of Hammett
vealed that from the three possible pathways (i) inverting
constants of meta substituents.
ester 2a gave poor results with Pisa1, presumably owing to
its hydrolytic instability at pH ≈ 8 going alongside compet-
ing spontaneous (non-enzymatic) hydrolysis, thereby erod-
ing the ee of product 2b.[8] By aiming to suppress the back-
ground hydrolysis by optimization of reaction conditions,
we initiated a detailed study on the mechanism of enzymatic
and non-enzymatic hydrolysis of sec-allylic and benzylic
sulfate esters rac-1a–8a. Although aryl and n-alkyl sulfates
have been thoroughly investigated regarding their stability
towards hydrolysis,[9,10] no detailed studies are available on
the hydrolysis of sec-alkyl sulfate esters. The majority of
investigations deal with detergents, such as sodium dodecyl
sulfate [11] or related anionic surfactants,[12] which predomi-
nantly consist of primary alkyl sulfates, in which the stereo-
chemical consequences of hydrolysis are not an issue. Stud-
ies on highly branched neopentyl sulfate reported re-
arrangement issues.[13]
Introduction
Enantioselective hydrolysis of ester and amide bonds cat-
alyzed by lipases, esterases, and proteases represents a land-
mark in biotransformations.[1] Their (industrial) application
was significantly widened by introduction of dynamic reso-
lution concepts that make use of in situ racemization[2] to
overcome the 50%-yield threshold of kinetic resolution. As
an alternative, simultaneous (or stepwise) transformation of
a pair of substrate enantiomers through stereochemically
opposite pathways leads to deracemization.[3] For the latter
concepts, hydrolytic enzymes acting through retention or
inversion of configuration are a crucial prerequisite.[4] In
this context, we recently developed a deracemization proto-
col for rac-sec-alcohols through enantio-complementary hy-
drolysis of their corresponding sulfate monoesters by using
a pair of sulfatases acting through stereo-complementary
pathways.[5] The key enzymes employed were the retaining
aryl sulfatase, PAS, from Pseudomonas aeruginosa[6] and the
inverting alkyl sulfatase, Pisa1, from Pseudomonas sp. DSM
6611.[7] Fortunately, Pisa1 displayed a very broad substrate
spectrum encompassing linear and branched sec-sulfate es-
ters that bear various functional groups, such as allylic C=C
and propargylic CϵC bonds, which are prone to undergo
side reactions with transition metal catalysts used in dy-
namic resolution protocols.[8] In contrast, benzylic sulfate
Results and Discussion
During our initial studies[8] we attempted to improve in-
complete stereoselectivities observed with several allylic,
propargylic and benzylic sec-sulfate esters by addition of
dimethyl sulfoxide (DMSO). Although positive effects were
observed, the exact molecular reason for this selectivity-en-
hancement – suppression of spontaneous (non-enzymatic)
hydrolysis and/or alteration of the catalytic properties of the
enzyme[14] – remained unknown.
[a] Department of Chemistry, Organic & Bioorganic Chemistry,
University of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
E-mail: Kurt.Faber@Uni-Graz.at
http://biocatalysis.uni-graz.at/
The influence of the polarity of water-miscible organic
co-solvents on the ee of 1b obtained from non-enzymatic
hydrolysis of enantiopure (S)-1a was investigated (Table 1).
Although significant racemization took place in neat buffer
[b] Department of Organic Chemistry, Graz University of
Technology,
Stremayrgasse 9, 8010 Graz, Austria
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402211.
N
[ee of (R)-1b 34%, ET of H2O ≈ 1], this effect gradually
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Hydrolysis of Benzylic sec-Sulfate Esters
diminished upon decreasing the polarity (as indicated by ilar effect (eeP 25% versus 52%, respectively). Both effects
N [15]
the Dimroth–Reichardt parameter ET
)
of the organic indicate the involvement of a polar (e.g. an allylic carb-
N
co-solvent used [ee of (R)-1b 48%, ET of DMSO 0.44]. enium ion) species.
Reducing the reaction temperature from 60° to 20 °C in
To support the hypothesis that a polar carbenium ion
Tris-buffer in the absence of organic co-solvent had a sim- species causes racemization during non-enzymatic hydroly-
sis, a series of benzylic sec-sulfate esters (2a–8a) were sub-
jected to non-enzymatic and enzymatic hydrolysis with
Pisa1 (Scheme 1, Table 2). Of special interest were the meta
substituted derivatives 2a–6a, because the electronic effects
of the meta substituents on the (de)stabilization of a benz-
ylic carbenium ion can be easily correlated to their Ham-
mett constants.[16] Substrate 7a was incorporated from ref.[8]
for comparison and pyridyl-analog 8a was used as an elec-
tron-deficient heterocyclic candidate.
Table 1. Non-enzymatic hydrolysis of (S)-1-octen-3-yl sulfate (1a)
in the presence of water-miscible organic co-solvents.[a]
Co-Solvent
ee of (R)-1b
Dimroth–Reichardt parameter
N [b]
[%]
(ET
)
Substrates rac-2a–8a were subjected to enzymatic hydrol-
ysis under standardized reaction conditions by using Pisa1,
the eeP of sec-alcohols (S)-2b–8b formed was determined by
GC analysis on a chiral stationary phase after extractive
separation from the remaining non-hydrolyzed sulfate esters
(S)-2a–8a. The latter were subjected to acid-catalyzed hy-
drolysis through strict retention of configuration[8] to yield
None
34
44
45
46
48
≈ 1
Methanol
Ethanol
2-Propanol
DMSO
0.76
0.65
0.55
0.44
[a] Conditions: Tris-buffer 100 mm, pH 8.0, 20% (v/v) co-solvent,
5 mg/mL (S)-1a, 90 h at 30 °C. [b] ET values are given for pure
solvents.
N
Scheme 1. Stereoselective hydrolysis of allylic and benzylic sec-sulfate esters by using inverting alkylsulfatase Pisa1.
Table 2. Enzymatic and non-enzymatic hydrolysis of benzylic sec-sulfate esters rac-2a–8a.
Substrate
Co-solvent[a]
Conversion [%][b]
eeP [%]
E[c]
Autohydrolysis [%][d]
σm constant[e]
rac-2a[f]
none
DMSO
none
DMSO
none
DMSO
none
DMSO
none
DMSO
none
DMSO
none
Ͼ 99
Ͼ 99
99
82
60
50
60
50
54
50
10
13
50
3.6
4.1
40
82
60
93
61
93
85
Ͻ 2
Ͻ 2
2.6
10
12
96
13
84
70
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 96
Ͼ 96
96
34
13
2
10
1
4
0.3
Ͻ 0.3
Ͻ 0.3
12
0.00
rac-3a
rac-4a
rac-5a
rac-6a
rac-7a[g]
rac-8a
0.12
0.34
0.37
0.43
n.a.
n.a.
99
Ͼ 99
Ͼ 99
99
DMSO
48
Ͼ 99
6
[a] Standard conditions: Pisa1 (0.13 mg), Tris-buffer, 100 mm, pH 8.0, substrate 2a–8a (5 mg/mL), 24 h at 30 °C; a 20% v/v. [b] Calculated
from eeS/(eeS + eeP). [c] Enantiomeric Ratio (E) calculated from eeP and eeS: E = {ln[(1-eeS)/(1 + eeS/eeP)]}/{ln[(1 + eeS)/(1 + eeS/eeP)]};[17]
for the application of E values to kinetic resolutions with competing autohydrolysis, see ref.[18] [d] Conversion in the absence of enzyme.
[e] Hammett constant of substituent R in the meta position (Scheme 1). [f] For data from 6 h reaction time, see ref.[8]. [g] For data from
72 h reaction time, see ref.[8]; n.a. = not applicable.
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M. Toesch, M. Schober, R. Breinbauer, K. Faber
FULL PAPER
corresponding alcohols (S)-2b–8b for ee-determination. Au- piece of cooperative acid-base catalysis:[7] Nucleophilic at-
tohydrolysis was measured under identical conditions in the tack of [OH–] onto C (derived from H2O by a binuclear
absence of enzyme. Absolute configurations were elucidated Zn2+ cluster in the active site of the enzyme) is comple-
by co-injection with authentic reference materials with mented by simultaneous protonation of the sulfate ester
–
known absolute configuration.[8] DMSO was selected as co- moiety through histidine 317 to generate HSO4 . All of
solvent because it showed the strongest selectivity-enhanc- these processes basically proceed through SN2 at C, because
ing effects (Table 1).
the generation of an aliphatic carbenium ion would be ener-
The enzymatic hydrolysis of substrate rac-2a was strongly getically too costly.
outcompeted by non-selective autohydrolysis and conse-
With benzylic substrates, such as 5a, a resonance-stabi-
quently gave alcohol 2b in near racemic form. Although the lized carbenium ion has to be taken into account, because
addition of DMSO showed a positive trend, the effects were clear correlation of the decrease of autohydrolysis with the
too small to be truly beneficial.
electron-withdrawing effects of meta substituents (as indi-
Introduction of electron-withdrawing substituents in the cated by their Hammett constants) strongly suggests that
meta position (substrates 3a–6a) gave increasingly better re- autohydrolysis (at least in part) occurs through an SN1-
sults, i.e. the gradual suppression of autohydrolysis gave a mechanism via an intermediate benzylic carbenium ion.
strong improvement in the apparent enantioselectivities[18] Our investigations on the mechanism of autohydrolysis was
from barely detectable (E = 2.6) to a respectable value (E = led by the following considerations: (i) analysis of the ee of
70). In line with the suppression of autohydrolysis, the over- formed alcohol 5b (and its potential erosion) derived from
all reaction rates slowed from 2a to 6a, indicated by enantiopure substrate (R)-5a would prove the existence of
decreasing conversion values. The addition of DMSO (20% a transient benzylic carbenium ion responsible for racemi-
v/v) further decreased autohydrolysis and hence gave even zation; (ii) use of 18O-labelled water would allow determi-
better overall enantioselectivities of up to E Ͼ 200. The cor- nation of the site of nucleophilic attack (S versus C)
relation between the electronic properties of the meta sub- through incorporation of [OH–] either into the formed
stituents, as denoted by their Hammett σm-values, is re- alcohol (attack at C) or into inorganic sulfate (attack at S)
markably strong: there is a drastic improvement in selectiv- to prove inversion or retention of configuration, respec-
ity owing to decreased autohydrolysis going from R = H tively (Scheme 2).
(0.00) through R = MeO (0.1) to R = Hal (Ͼ 0.3), whereas
both halo-derivatives with comparable σm-values of 0.34
and 0.37 gave similar results. A further significant improve-
ment was achieved with the CF3-derivative (0.43).
The beneficial effect of electron-deficient substituents in
the meta position is nicely underlined by doubly meta sub-
stituted substrate 7a, which could be resolved with perfect
enantioselectivity.[8] To test whether this electronic effect
could also be extended to heteroaromatic benzylic analogs,
electron-deficient 3-pyridyl derivative 8a was investigated.
In line with the above trends, it could be resolved with ex-
cellent results (E Ͼ 200). Unfortunately, attempts to synthe-
tize electron-rich derivatives, such as 1-(furan-2yl)ethyl sulf-
ate, 1-(thiophen-2-yl)ethyl sulfate, 1-(1H-pyrrol-2-yl)ethyl
sulfate or imidazole analogs, which could serve as
counterproof, were unsuccessful owing to the instability of
the corresponding sec-alcohols.
Scheme 2. Elucidation of retaining (SN2 at S), inverting (SN2 at C)
The hydrolysis of sec-alkyl monosulfate esters is a com-
and racemizing (SN1) pathways of non-enzymatic and enzymatic
plex process: Acid catalysis proceeds by protonation of the
hydrolysis of (R)-5a through 18O-labeling (k values are stated as
negatively charged sulfate ester moiety[19] at the C–O–S
first order relative rate constants).
bridge atom, which allows nucleophilic attack of H2O at
sulfur, along with release of the alcohol and HSO4 as a
–
To check the validity of the method, enzymatic hydrolysis
good leaving group.[20] Consequently, it is a fast process and of (R)-5a [enantiomer ratio (e.r.) Ͼ99:Ͻ1] by using inverting
proceeds with retention of configuration at the chiral C- Pisa1 in 18OH2 was performed as a control experiment.[7]
atom bearing the sulfate ester moiety. However, nucleo- For handling purposes, the medium was composed of 16O-
philic attack of [OH–] at C under basic conditions would Tris-buffer (0.1 mL, 1 m, pH 8.0) diluted at a ratio of 1:10
proceed through inversion at C, but it is hardly possible, with 18O-labelled H2O (label 97:3). Addition of Pisa1
because the approach of [OH–] onto the negatively charged (2.6 mg) from 4.6 μL of 16OH2 stock solution led to a (cal-
substrate is disfavored and the process would generate culated) 18/16O-ratio in the reaction medium of 84:16. After
2–
SO4 as a poor leaving group; hence, it is an exceedingly 24 h of reaction time, analysis of alcohol 5b by GC–MS
slow process.[21,22] In contrast, the enzymatic hydrolysis as with a chiral stationary phase revealed an e.r. of Ͼ99:Ͻ1
exemplified by inverting alkylsulfatase Pisa1 is a master- for the (S)-enantiomer with an 18/16O-label of 79:21. These
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Eur. J. Org. Chem. 2014, 3930–3934

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
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M. Toesch, M. Schober, R. Breinbauer, K. Faber
FULL PAPER
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Received: March 6, 2014
Published Online: May 6, 2014
Chemistry, 4th ed., Wiley-VCH, Weinheim, Germany, 2010.
3934
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 3930–3934