Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase

Article abstract of DOI:10.1002/anie.201713189

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols.

Full text of DOI:10.1002/anie.201713189

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Accepted Article
Title: Kinetic Resolution of sec-Thiols via Enantioselective Oxidation
with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase
Authors: Mathias Pickl, Alexander Swoboda, Elvira Romero, Christoph
Winkler, Claudia Binda, Andrea Mattevi, Kurt Faber, and
Marco Fraaije
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201713189
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10.1002/anie.201713189
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Kinetic Resolution of sec-Thiols via Enantioselective Oxidation
with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase
Mathias Pickl, Alexander Swoboda, Elvira Romero, Christoph K. Winkler, Claudia Binda, Andrea
Mattevi, Kurt Faber,* and Marco W. Fraaije*
Abstract: Various flavoprotein oxidases were recently shown to
oxidize prim-thiols. Here we extend this reactivity towards sec-thiols
via structure-guided engineering of 5-(hydroxymethyl)furfural oxidase
(HMFO). The variants obtained were employed for the oxidative
kinetic resolution of rac-sec-thiols yielding the corresponding thio-
ketones and nonreacted (R)-thiols with excellent enantioselectivities
(E ≥ 200). The engineering strategy applied went beyond the classic
approach of replacing bulky amino acid residues with smaller ones,
as the active site was additionally enlarged by a newly introduced Thr
residue that establishes a hydrogen bonding interaction with the
substrates, as verified in the crystal structure of the variant. These
strategies unlocked HMFO variants for the enantioselective oxidation
of a range of sec-thiols.
methods, such as the kinetic resolution of thiols with baker’s yeast
were only moderately successful.[6] The prominent lipase B from
Candida antarctica catalyzes the enantioselective hydrolysis of
thioesters (Scheme 1a), in contrast, the kinetic resolution via
esterification of thiols failed.[7]
The demand for chiral building blocks for pharmaceutical appli-
cations is a driving force for the development of new methodo-
logies in modern synthetic chemistry. In this context, biocatalysis,
which excels in producing chiral molecules, represents an
increasingly important tool for synthetic chemists.[1] Organosulfur
moieties constitute highly relevant structural elements with a
broad range of bio-activities, and efficient strategies for their
selective production are sought. Examples include the acyl-coA:
cholesterol acyltransferase inhibitor Eflucimibe, the leukotriene
receptor antagonist Montelukast, or the calcium channel blocker
Diltiazem (Figure 1).[2] Recent approaches for the synthesis of
nonracemic sec-thiols include the ring-opening of chiral thiazol-4-
ones produced by organo-catalytic Michael addition,[3] S-H bond
insertion cooperatively catalyzed by a rhodium catalyst and a
chiral spirophosphoric acid,[2f] and the formation of chiral vic-
hydroxythiols from cis-epoxides.[4] However, these systems suffer
from the requirement of complex starting materials and/or high
loadings of sophisticated organocatalysts. For the kinetic reso-
lution of racemic thiols via esterification, organo- and biocatalytic
methods are known. Cinchona alkaloid-based organocatalysts
allowed the resolution of various benzylic sec-thiols, though
excellent enantioselectivities were achieved only with a single
substrate at high catalyst loadings (Scheme 1a).[5] Biocatalytic
Figure 1. Examples of active pharmaceutical ingredients containing sec-thiol
moieties.
Flavoprotein oxidases, which are well established for the
dioxygen-dependent oxidation of alcohols to carbonyl moieties,[8]
were recently shown to catalyze the transformation of prim-thiols
into the corresponding thiocarbonyl analogues.[9] One of the
enzymes exhibiting this activity is 5-(hydroxymethyl)furfural
oxidase (HMFO; EC 1.1.3.47).[10] In addition to its ability to
produce the polymer building-block 2,5-furandicarboxylic acid
from 5-(hydroxylmethyl)furfural,[11] HFMO is able to oxidize a
broad range of electronically activated benzylic and cinnamic
prim-alcohols,[10] and its variants transform also sec-alcohols in a
stereo-preferred fashion.[12] The enzyme oxidizes the alcohol (or
the hydrated aldehyde) to the corresponding carbonyl/carboxyl
product by formal hydride abstraction via oxidized flavin (FAD).
FADH2 thus formed reduces molecular oxygen forming hydrogen
peroxide as byproduct.
The utility of HMFO variants for sec-alcohol-oxidation on the
one hand and wild-type activity on prim-thiols on the other hand
triggered our interest in designing HMFO variants that would
combine these two features yielding an enzyme being active on
sec-thiols. Such a biocatalyst would enable the production of non-
racemic sec-thiols at the expense of molecular oxygen through
enantioselective oxidation. According to the (S)-stereo-
preference of HMFO variants for sec-alcohols, (R)-sec-thiols
could be expected as unreacted enantiomers together with the
corresponding thioketones (Scheme 1b).
[a]
[b]
Dr. E. Romero, Prof. Dr. M. W. Fraaije
Department of Biotechnology, University of Groningen
Nijenborgh 4, 9747AG, Groningen (The Netherlands)
E-mail: m.w.fraaije@rug.nl
M.Sc. M. Pickl, A. Swoboda, Dr. C. K. Winkler, Prof. Dr. K. Faber
Department of Chemistry, University of Graz
Heinrichstrasse 28, A-8010, Graz (Austria)
E-mail: kurt.faber@uni-graz.at
[c]
[d]
Dr. C. K. Winkler
Austrian Centre of Industrial Biotechnology (ACIB GmbH)
c/o Heinrichstrasse 28, A-8010 Graz (Austria)
Dr. C. Binda, Prof. Dr. A. Mattevi
Department of Biology and Biotechnology “Lazzaro Spallanzani”
University of Pavia
Via Ferrata 9, 27100, Pavia (Italy)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201713189
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In addition, in silico models suggested that the mutation of
Val465 to Ser or Thr would provide additional space by rotating
their side chain to establish a hydrogen bond with His467 and/or
Glu306.[12a] To validate this hypothesis, we determined the crystal
structure of the Val465Thr protein (Figure 2). The overall structure
of the mutant enzyme is essentially unaltered with respect to wild-
type protein (rmsd is 0.49 Å for the 529 Cα atoms of the two
monomers present in the asymmetric unit). The only exception is
represented by two loops that in monomer B lack clear electron
density (residues 99-104 and 203-209). The active site
architecture is also highly conserved except for the mutation site
(Figure 2b). Inspection of the electron density revealed that
Thr465 adopts a double conformation, one being identical to that
of Val465 in the wild-type enzyme and another one displaying a
rotamer conformation that establishes a H-bond with Glu306
(Figure 2b-c). Similarly to 1-phenylethanol which was previously
modeled in the crystal structure of the wild-type enzyme,[12a] 1-
phenylethanethiol would perfectly fit in the mutant active site
because the alternative conformation of Thr465. These findings
validated the in silico modeling prediction and prompted further
analysis of Val465Thr and Val465Ser mutants.
In previous studies, commonly used oxidase assays could be
used to determine HMFO activities.[9, 12a] However, these assays
were unsuitable for the slower reacting sec-thiols studied in the
present work. As alternative, the pseudo-first-order rate constants
Scheme 1. Methods for kinetic resolution of sec-thiols.
for anaerobic flavin reduction under saturation conditions (kred
)
were determined using a stopped-flow apparatus. This allowed us
to estimate the overall activity, because hydride transfer in the
reductive half-reaction was demonstrated to be the rate-limiting
step.[12b, 12c] Furthermore, the effect of mutations on substrate
binding was simultaneously studied by determining the apparent
macroscopic dissociation constant for binding of the substrate to
the enzyme (Kd). For all stopped-flow measurements, the model
substrate rac-1a was used (Scheme 2). Whereas wild-type HMFO,
Trp466His HMFO, and Trp466Ala HMFO did not show any
detectable activity with rac-1a, all other variants exhibited
significant flavin reduction (Table 1). Remarkably, the activities
and Kd values were in a similar range. An attempt to combine both
space-creating effects in a double mutant Trp466Phe/Val465Thr
failed: no additional improvement over the corresponding single
The geometry of the active site of wild-type HMFO explains
the distinct selectivity for prim- over sec-alcohols.[12a] The sub-
strate positioning is mainly guided by H-bonding of His467 with
the substrate hydroxyl group. This arrangement allows the
abstraction of the pro-R hydrogen at the substrate Cα atom by the
flavin N5 atom.[12] In order to provide additional space for the
methyl group of sec-alcohols, in a previous study Trp466 was
mutated.[12a] In the present work, Trp466 was replaced by smaller
amino acids in order to create a more spacious active site for sec-
thiols: Trp466Phe, Trp466Ala,Trp466Tyr and Trp466His mutants
were created. The latter two mutations were chosen in view of the
beneficial effect on dioxygen reactivity of an aromatic residue at
the 466-position as found in the structurally related aryl-alcohol
oxidase (EC 1.1.3.7).[12b]
Figure 2. Crystal structure of the Val465Thr HMFO mutant. (a) The overall structure is shown as ribbon representation embedded in a semi-transparent surface in
a clipped view to highlight the cleft space of the active site. The FAD cofactor is depicted sticks with carbon atoms in yellow. (b) Zoomed view of the Val465Thr
HMFO active site showing the Thr465 double conformation. The alternative conformation of Thr465 is in black (H-bond with Glu306 is represented as dashed line).
The (S)-1-phenylethanethiol substrate was modelled in the enzyme active site (carbon atoms in magenta). The orientation of the molecule is approximately 180°
rotated along a vertical axis with respect to (a). (c) The weighted 2Fo-Fc electron density map, contoured at 1.2 σ level, around the double conformation of Thr465.
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10.1002/anie.201713189
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mutants could be generated. Overall, the strategy to introduce
activity on sec-thiols was successful, as the Kd of rac-1a for all
active variants was comparable to that of benzyl alcohol with wild-
type HMFO (Kd = 1.7 mM).[12b, 12c]
excellent enantioselectivity. Overall, unreacted (R)-enantiomers
were recovered for all substrates in isolated yields of 15-61% after
column chromatography. The absolute configuration of 1a, 1c, 1g
and 1h was determined to be (R) by comparison of optical rotation
values with literature data.[5,19] The absolute configuration of 1b
and 1d-f was concluded to be (R) due to the contiguous (+)-sense
of optical rotation of 1a-h, which is in line with identical elution
order on chiral HPLC [(R) first, (S) second, see ESI]. The absolute
configuration for 1e was additionally confirmed by CD
spectroscopy (Figure S8). Overall, the Val465Ser HMFO variant
proved to be a reliable biocatalyst for the kinetic resolution of
benzylic sec-thiols.
Motivated by the promising kinetic properties observed for the
above HMFO variants, their biocatalytic applicability was
investigated with substrate rac-1a. DL-dithiothreitol (DTT) was
added as anti-oxidant to the reaction mixture (Figure S1 in the
ESI).[13] This suppressed the formation of disulfide adducts and
traces of the elimination product styrene for all tested substrates.
Confirming the kinetic data, wild-type HMFO was almost un-
reactive with rac-1a (~3% conversion). In contrast, all variants
gave good conversions in the oxidation of rac-1a (up to 42%,
Figure 3) and an enantiomeric excess of substrate (ees) of up to
91% (Table 2). The degrees of conversion correlate well with the
determined kinetic parameters (Figure 3 and Table 1). The
highest conversions were obtained with the two Val465 mutants
which also displayed the lowest Kd values. No conversion of the
substrate was observed in control reactions without enzyme.
Overall, Val465Ser was identified as the most potent variant and
was used for follow-up experiments.
Table 1. Pre-steady-state kinetic parameters of HMFO variants on rac-1-
phenylethanethiol (1a).[a]
HMFO
kred [s-1]
Kd [mM]
n.d.
Wild-type
<0.001
Trp466His
Trp466Ala
Trp466Phe
Trp466Tyr
Val465Ser
Val465Thr
Val465Thr/Trp466Phe
<0.001
n.d.
<0.001
n.d.
0.034 ± 0.007
0.010 ± 0.001
0.020 ± 0.002
0.021 ± 0.003
0.009 ± 0.003
1.3 ± 0.5
1.1 ± 0.3
0.6 ± 0.1
0.6 ± 0.2
1.3 ± 0.9
Figure 3. Conversion of rac-1a by HMFO-variants. Reactions were performed
in phosphate buffer (100 mM, pH 7.0) containing isopropanol (1%, v/v), DTT (50
mM), 5 mM substrate, and HMFO variants (3.5 µM), at 30 °C for 24 h in air-
saturated solutions (235 µM dioxygen). Disulfide formation <3 %.
The formation of thioacetophenone (2b) as oxidation product
of rac-1a was proven by GC-analysis using reference material 2b
independently synthesized using Lawesson’s reagent (Figure S4
in the ESI),[15] confirming previous observations.[9] However, the
latter are rapidly hydrolyzed via monothioketals in aqueous
medium yielding the corresponding carbonyl analogues going in
hand with formation of hydrogen sulfide. Consequently, the cor-
responding acetophenone derivatives 3a-h were predominantly
found in the reaction mixture after 24 h (Figure S5 in the ESI).[16]
Attempts to convert the kinetic resolution into a cyclic
deracemization[17] through (chemical) in-situ reduction of the thio-
ketone formed in analogy to the cyclic deracemization of amines
employing amino acid[19] or amine oxidases[20] and boron-based
reducing agents, unfortunately failed: neither borohydrides nor
borane complexes were reactive enough to compete with the fast
spontaneous hydrolysis of the thioketone intermediate (see ESI).
In summary, rationally designed variants of HMFO were
employed for the enantioselective oxidation of 1-phenylethane-
thiols, which allowed their kinetic resolution with unprecedented
selectivities (E up to > 200). Variants were created by not only
replacing bulky amino acid residues with smaller ones, but also
by a space-creating rearrangement of amino acids within the
active site enforced by additional H-bonding, which was predict
[a] Experiments were performed at 30 °C in phosphate buffer (100 mM, pH
7.0) under anaerobic conditions. Reduction was measured at Amax of HMFO-
variant (See ESI, Table S2). n.d. = not detectable.
In order to prove the general applicability of this method, the
tolerance of electron-donating and -withdrawing substituents on
the aromatic ring were evaluated since the electron density on the
aromatic ring may influence the rate of hydride transfer in
HMFO.[14] m- and p-substituted substrates rac-1b-g and the
naphthyl analogue 1h were tested (Scheme 2). The latter were
prepared from the corresponding alcohols via mesylation by the
Mitsunobu protocol, followed by nucleophilic substitution with thio-
acetate and thioester reduction over lithium aluminium hydride
(see ESI).[5]
HMFO-Val465Ser
accepted
all
1-phenylethanethiol
analogues (Table 2). Rac-1f and rac-1g, bearing activating
electron-donating substituents were converted in up to 50% with
ees of >97% for (R)-enantiomers, which corresponds to perfect E-
values of >200. On the contrary, the conversion of bromo-
substituted substrates rac-1d and rac-1e was slower, albeit with
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10.1002/anie.201713189
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COMMUNICATION
Scheme 2. Biocatalytic enantioselective oxidation of (S)-1-phenylethanethiols 1a-h with HMFO-variants.
column chromatography on silica gel and the ee of (R)-thiols was
measured on chiral HPLC after derivatization (see SI).
Table 2. Enantioselective oxidation of 1-phenylethanethiol derivatives by
Val465Ser HMFO variant.[a]
rac-Substrate
Conversion
[%][b]
ees (R) [%][c]
E[d]
Acknowledgements
1a
1b
1c
1d
1e
1f
48
91
>200
45
Funding by the Austrian Science Fund (FWF) within the DK
Molecular Enzymology (project W901) and the Austrian BMWFW,
BMVIT, SFG, Standortagentur Tirol, Government of Lower
Austria and ZIT through the Austrian FFG-COMET-Funding
Program is gratefully acknowledged. The authors wish to thank
Doris Loidolt for technical assistance and Philipp Neu and Walter
Keller for HR-MS and CD-measurements.
50
88
47
87
>200
>200
150
44 (50)[e]
30 (45)[e]
50
78 (>97)[e]
42 (80)[e]
>97
>200
>200
>200
Keywords: alcohol oxidase • biocatalysis • enzyme engineering
• kinetic resolution • sec-thiol oxidation
1g
1h
49
>97
44 (49)e
78 (93)e
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COMMUNICATION
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COMMUNICATION
Mathias Pickl, Alexander Swoboda,
Elvira Romero, Christoph K. Winkler,
Claudia Binda, Andrea Mattevi, Kurt
Faber,* and Marco W. Fraaije*
Page No. – Page No.
Kinetic Resolution of sec-Thiols via
Enantioselective Oxidation with
Rationally Engineered 5-
An alcohol oxidase as a tool for the preparation of nonracemic sec-thiol
building blocks: Rationally designed variants of 5-(hydroxymethyl)furfural (HMF)
oxidase catalyse the enantioselective oxidation of 1-phenylethanethiols, achieving
their kinetic resolution with unprecedented selectivities. The crystal structure of one
of the variants explains this unique finding.
(Hydroxymethyl)furfural Oxidase
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