One-pot deracemization of sec-alcohols: Enantioconvergent enzymatic hydrolysis of alkyl sulfates using stereocomplementary sulfatases

Article abstract of DOI:10.1002/anie.201209946

Hand in hand: The title transformation was achieved using a pair of sulfatases acting through inversion and retention of configuration on opposite substrate enantiomers. Using Pseudomonas aeruginosa arylsulfatase PAS with alkylsulfatase PISA1 in one-pot l

Full text of DOI:10.1002/anie.201209946

Angewandte
Chemie
DOI: 10.1002/anie.201209946
Biotransformations
One-Pot Deracemization of sec-Alcohols: Enantioconvergent
Enzymatic Hydrolysis of Alkyl Sulfates Using Stereocomplementary
Sulfatases**
Markus Schober, Michael Toesch, Tanja Knaus, Gernot A. Strohmeier, Bert van Loo,
Michael Fuchs, Florian Hollfelder, Peter Macheroux, and Kurt Faber*
Given the fact that the theoretically possible number of
racemates is larger than that of symmetric prochiral or
meso compounds,[1] the development of deracemization
methods, which yield a single stereoisomer from a racemate
is an important topic.[1–3] Enantioconvergent processes are
based on the transformation of a pair of enantiomers through
opposite stereochemical pathways affecting retention and
inversion of configuration. Depending on the stereochemical
course of enzymatic and chemical reactions, three types of
deracemization protocols were recently classified by Feringa
et al.[4] Two chemoenzymatic methods start with a biocatalytic
kinetic resolution step, which yields a hetero- or homochiral
1:1 mixture of the formed product and nonconverted sub-
strate enantiomer. The latter is subjected to a second (non-
enzymatic) transformation with retention or inversion of
configuration to yield a single stereoisomeric product.
Although several one-pot, two-step protocols have been
successfully demonstrated,[5,10c,d] they typically rely on acti-
vated species, such as sulfonates,[5a–d] nitrate esters,[5b] or
Mitsunobu intermediates,[5e] and negatively affect the overall
atom economy of the process. The most elegant method relies
on one (or two) enzyme(s), which mediate the transformation
of both enantiomers through stereocomplementary pathways
by retention and inversion. Since the requirements of such
double selectivities are very difficult to meet, successful
examples are rare: This approach has been applied to the
hydrolysis of epoxides using two epoxide hydrolases showing
opposite enantiopreference[6] or a single enzyme that cata-
lyzes the enantioconvergent hydrolysis of enantiomers with
opposite regioselectivity.[7]
For enzymes, the ability to act by retention or inversion is
a rare feature, which has been found among epoxide hydro-
lases,[8] dehalogenases,[4,9] and sulfatases.[10] The latter catalyze
the hydrolytic cleavage of (alkyl) sulfate esters by breakage of
ꢀ
ꢀ
the S O or the C O bond leading to retention or inversion at
the chiral carbon atom,[10b] and thus makes them prime
candidates for enantioconvergent processes. So far, only
a single inverting sec-alkylsulfatase (PISA1) was generated
recombinantly and characterized biochemically,[11] thus allow-
ing preparative-scale applications.[10c] In combination with
acid-catalyzed hydrolysis of the nonreacted substrate enan-
tiomer under retention of configuration[12] a chemoenzymatic
two-step deracemization protocol for sec-alcohols was
recently developed.[10c,d] However, the method suffers from
serious limitations because it requires undesirably large
volumes organic solvents and several molar equivalents of
a strong acid (typically 2–7 equiv of p-TosOH), which pose
the risk of racemization or decomposition to the functional-
ized substrates, especially when elevated temperatures are
required for acidic hydrolysis. Moreover, it is not applicable to
retaining sulfatases, because no chemical method for sulfate
ester hydrolysis with inversion exists.[10c]
[*] M. Schober, M. Toesch, Dr. M. Fuchs, Prof. K. Faber
Department of Chemistry, Organic & Bioorganic Chemistry,
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: kurt.faber@uni-graz.at
So far, retaining-sulfatase activity was reported in whole
cells of Rhodopirellula baltica DSM 10527,[13] but the
corresponding enzymes could not be identified, thus imped-
ing the use of recombinant technology to make the enzyme
available for biocatalysis. Furthermore, the retaining sulfatase
of Rh. baltica would not be suitable for an enantioconvergent
process with PISA1, because both proteins exhibit the same
enantiopreference. During our search for a retaining sec-
alkylsulfatase with an enantiopreference opposite to that of
PISA1, we discovered that the arylsulfatase from Pseudomo-
nas aeruginosa (PAS) exhibited activity on sec-alkylsulfates.
PAS, which has been characterized on a molecular level,[14]
showed promiscuous activity on various arylic phosphates and
phosphonates.[15] On its standard model substrate (4-nitro-
phenyl sulfate), PAS exhibited a rate acceleration of kcat/kuncat
2.3 ꢀ 1010,[16] and for a less reactive substrate the highest rate
enhancement (kcat/kuncat = 2 ꢀ 1026) of any catalytic reaction
known so far has been measured.[17] The stereochemical
Homepage: http://biocatalysis.uni-graz.at/
Dr. T. Knaus, Prof. P. Macheroux
Institute of Biochemistry, Graz University of Technology
Dr. G. A. Strohmeier
ACIB GmbH c/o Department of Organic Chemistry
Graz University of Technology
Dr. B. van Loo, Prof. F. Hollfelder
Department of Biochemistry, University of Cambridge
[**] This study was financed by the Austrian Science Fund within the DK
Molecular Enzymology (FWF, project W9), the BMWFJ, BMVIT,
SFG, Standortagentur Tirol, and ZIT through the COMET-Program.
F.H. and B.v.L. were supported by the BBSRC and F.H. as an ERC
Starting Investigator. The authors would like to thank Barbara
Grischek, Sebastian Grimm, Gerald Rechberger, and Nina Schmidt
for their valuable assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209946.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://angewandte.org/open.
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ference with inversion[10c,d] of PISA1,[10e] we optimized the
enzymatic hydrolysis of the substrates 1a–7a with the latter
enzyme (Table 1). Most of the substrates (1a, 4a–7a) showed
perfect E values of greater than 200, with only 2a and 3a
giving insufficient selectivities. The data thus obtained
enabled us to develop three types of enantioconvergent
processes (Scheme 1, Table 2):
Table 1: Kinetic resolution of sulfate esters rac-1a–7a with retention and
inversion using PAS and PISA1.
Substrate Enzyme t [h] Conv. eeP [%]
eeS [%]
E value[a]
[%][a]
rac-1a[b]
rac-2a
PAS
PAS
PAS
PAS
PAS
PAS
PAS
24
24
6
6
6
6
48
6
4
6
49
49
46
61
53
65
30
47
56
57
50
50
50
49
97 (S)
91 (S)
98 (S)
48 (S)
11 (S)
22 (R)
>99 (S)
98 (S)
55 (S)
32 (S)
92 (R)
89 (R)
84 (R) >200 (S)
74 (R) 6 (S)
11 (R) <2 (S)
40 (S) ꢁ2 (R)
46 (R) >200 (S)
89 (S) >200 (S)
190 (S)
59 (S)
rac-3a
rac-4a[c
rac-5a[c]
rac-6a[c]
rac-7a
Scheme 1. One-pot, two-enzyme deracemization process using retain-
ing PAS and inverting PISA1.
rac-1a[b,d] PISA1
Table 2: Deracemization of the sulfate esters rac-1a–7a using retaining
PAS and inverting PISA1.
rac-2a[d]
rac-3a[d]
rac-4a[c]
rac-5a[c,e]
rac-6a[c,d]
rac-7a
PISA1
PISA1
PISA1
PISA1
PISA1
PISA1
80 (S)
40 (S) ꢁ3 (S)
8 (S)
Substrate
Reaction
Type
tPISA1 [h]
tPAS [h]
Conv.
[%]
eeP [%]
72
24
24
72
>99 (R) >99 (R) >200 (R)
>99 (R) >99 (R) >200 (R)
>99 (R) >99 (R) >200 (R)
rac-1a
rac-1a[a]
rac-1a[a,b]
rac-2a
rac-3a
rac-4a
rac-5a
rac-6a
rac-7a
C
C
C
A
A
B
B
B
C
24
24
24
12
6
72
48
48
72
24
24
24
36
18
24
24
24
72
93
98
93
93 (S)
95 (S)
98 (S)
91 (S)
97 (S)
>99 (S)
93 (S) >200 (S)
See the Supporting Information for reaction conditions. [a] Calculated
from eeP (ee value of product) and eeS (ee value of starting material)
according to Ref. [10c,d]. [b] 20% DMSO (v/v) as cosolvent to suppress
non-enzymatic background hydrolysis. [c] Switch in Cahn–Ingold–Prelog
priorities. [d] See Ref. [10c]. [e] See Ref. [10d].
81
>99
>99
>99
97
94 (R)
>99 (R)
>99 (R)
>99 (S)
80
features of PAS were investigated using a series of sec-
alkylsulfate esters (rac-1a–7a; Table 1). The substrates 1a–3a
bearing an acetylenic moiety on the long chain adjacent to the
stereocenter were resolved with good to excellent enantiose-
lectivities (E 59 to > 200). Undesired non-enzymatic back-
ground hydrolysis of 1a could be suppressed by addition of
20% (v/v) of DMSO as a cosolvent.[18] In contrast, the
selectivities were largely lost when the acetylenic moiety was
moved to the short chain (substrates 4a–6a). The alkyl aryl
derivative 7a gave again an excellent E value of greater than
200. All substrates converted with high enantioselectivities
(1a–3a, 7a) were hydrolyzed with complete retention of
configuration, thus yielding S-configured alcohols and
unreacted R-configured sulfate esters. To prove the stereo-
chemical course of sulfate ester hydrolysis by PAS, enzymatic
cleavage of rac-1-octyn-3-yl sulfate (6a) was performed in an
18O-labeled buffer (label > 98%). GC/MS analysis of the
alcohol 6b formed revealed that (in contrast to inverting sec-
alkylsulfatases[10c]) no incorporation of 18O occurred, and is
consistent with the attack of the enzymeꢁs formylglycine
nucleophile on sulfur. Hydrolysis of enantiopure (S)-6a by
PAS yielded alcohol (S)-6b in greater than 99% ee, thus
proving that hydrolysis proceeded under strict retention of
configuration.
See the Supporting Information for reaction conditions. [a] Double
enzyme concentrations. [b] Cosolvent 20% (v/v) DMSO.
*
Type A. The substrates rac-2a and rac-3a, where PAS was
highly enantioselective, could be deracemized by a one-
pot, two-step sequence using retaining PAS first, followed
by non-enantioselective inverting hydrolysis with PISA1
to yield (S)-2b and (S)-3b in 91 and 97% ee, respectively.
*
Type B. For rac-4a–6a, where PISA1 was highly enantio-
selective, but PAS was not, the opposite order of events—
PISA1 first, PAS second—was successful and yielded the
corresponding R-configured alcohols 4b–6b in 94 to
greater than 99% ee
*
Type C. The ideal single-step process using both enzymes
simultaneously was designed for the substrates rac-1a and
rac-7a. To maximize the ee value of 1b, DMSO was used as
cosolvent to suppress background hydrolysis which
increased the ee value of (S)-1b from 93 to 98% ee. To
demonstrate the applicability of this method on a prepara-
tive scale, the deracemization of rac-6a was scaled-up (1 g,
4.4 mmol), and gave (R)-6b as the sole product in 82%
yield upon isolation with 98% ee.
Since the stereochemical features of PAS—R enantiopre-
ference with retention—would ideally complement the S pre-
The choice of which process (Type A–C) is most suitable
for the deracemization of a given substrate depends on the
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[5] a) W. Szymanski, R. Ostaszewski, Tetrahedron: Asymmetry
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with retention or inversion of configuration. Processes of
Types A and B are feasible with a single enantioselective
enzyme, whereas Type C requires two enantioselective sulfa-
tases with matching opposite enantiopreference. It should be
kept in mind that Types A and B constitute kinetic reso-
lutions,[19] whereas Type C represents a parallel kinetic
resolution.[20]
Overall, the purely enzymatic protocol excels by its
significantly broader applicability compared to the chemo-
enzymatic procedure[10c,d] for the following reasons: 1) it
eliminates the harsh reaction conditions required for acid-
catalyzed hydrolysis, which are incompatible with sensitive
functional groups and 2) it is also applicable to retaining
sulfatases (such as PAS).
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In summary, the one-pot deracemization of sec-alcohols
bearing various functional groups was achieved by enantio-
convergent hydrolysis of the corresponding sulfate esters
using the retaining aryl sulfatase PAS and the inverting alkyl
sulfatase PISA1, which possess the required opposite enan-
tiopreference.
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[18] The cosolvent has no significant influence on the pH value and
does not hamper the workup procedure, but reduces the water
activity (aW) of the system, thereby impeding spontaneous (non-
enzymatic) hydrolysis.
[19] For the kinetis of two-step deracemizations, see: S. Pedragosa-
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Experimental Section
Preparative scale one-pot, one-step deracemization of rac-6a:
Purified PISA1 (0.5 mL, 13 mg, 176.5 nmol, 0.5 mL of stock solution)
was added to 1-octyn-3-yl sulfate (rac-6a, 1 g, 4.4 mmol) dissolved in
Tris-HCl (197.5 mL, 100 mm, pH 8.0). The reaction was shaken at
120 rpm and 308C for 24 h. PAS (N-terminal strep tag, 2 mL, 26 mg,
434 nmol) was added. After an additional 24 h, the aqueous phase was
extracted with tBuOMe (3 ꢀ 100 mL). The combined organic phases
were dried with anhydrous Na2SO4 and filtered. The solvent was
evaporated under reduced pressure (220 mbar, 308C) and (R)-6b was
obtained as a clear yellow oil (0.45 g, 3.6 mmol, 82%) with the
following physical properties: ee value 98% [determined via GC as
(R)-1-octyn-3-yl acetate]; ½aꢂD ¼ + 4.68 (CHCl3, c = 1.0); lit.[21]
20
20
1
½aꢂD ¼ + 5.38 (CHCl3, c = 1.0); H NMR (300 MHz, CDCl3): d = 4.39
(dt, 29.0 and 9.2 Hz, 1H), 2.48 (d, 5.2 Hz, 1H), 1.80–1.67 (m, 2H),
1.53–1.25 (m, 6H), 0.92 ppm (t, 6.1 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d = 85.0, 72.8, 62.3, 37.6, 31.4, 24.7, 22.5, 14.0 ppm.
Received: December 12, 2012
Revised: January 12, 2013
Published online: February 10, 2013
Keywords: alcohols · biotransformations · enzyme catalysis ·
.
kinetic resolution · synthetic methods
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