Access to lactone building blocks via horse liver alcohol dehydrogenase-catalyzed oxidative lactonization

Article abstract of DOI:10.1021/cs400535c

The oxidative lactonization of 1,4-, 1,5-, and 1,6-diols using horse liver alcohol dehydrogenase (HLADH) is reported. Molecular oxygen was used as terminal electron acceptor by utilization of the laccase-mediator concept to regenerate the oxidized nicotinamide cofactor and producing water as sole byproduct. Spontaneous hydrolysis of the lactone products was identified as a major limiting factor toward preparative application of the system, which can be alleviated by using a two liquid phase approach to extracting the product into an organic solvent.

Full text of DOI:10.1021/cs400535c

Letter
pubs.acs.org/acscatalysis
Access to Lactone Building Blocks via Horse Liver Alcohol
Dehydrogenase-Catalyzed Oxidative Lactonization
†
‡
†
‡
‡
Selin Kara, Dominik Spickermann, Joerg H. Schrittwieser, Andrea Weckbecker, Christian Leggewie,
†
,†
Isabel W. C. E. Arends, and Frank Hollmann*
†
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
‡
evocatal GmbH, Merowingerplatz 1a, 40225 Du
̈
sseldorf, Germany
*
S Supporting Information
ABSTRACT: The oxidative lactonization of 1,4-, 1,5-, and 1,6-diols using horse liver
alcohol dehydrogenase (HLADH) is reported. Molecular oxygen was used as terminal
electron acceptor by utilization of the laccase-mediator concept to regenerate the oxidized
nicotinamide cofactor and producing water as sole byproduct. Spontaneous hydrolysis of
the lactone products was identified as a major limiting factor toward preparative application
of the system, which can be alleviated by using a two liquid phase approach to extracting the
product into an organic solvent.
KEYWORDS: lactones, alcohol dehydrogenase, laccase-mediator system, cofactor regeneration, two liquid phase systems, biocatalysis,
chemoenzymatic cascades
actones represent an important class of substances with
In a first set of experiments we evaluated a range of 1,4-, 1,5-,
and 1,6-diols (Table 1). 1,3-Diols were evaluated as well but did
not yield detectable lactone formation, most probably because
of the significant ring strain of these systems. It is also worth
mentioning here that in the absence of any catalytic component
(HLADH, Mtlaccase, NAD+ or mediator) no significant
conversion was detected.
1
L
applications not only in polymer synthesis but also as
2
3
environmentally benign solvents, fuels, and as building blocks
4
for synthesis.
5
Their preparation via esterification of hydroxy acids or
6
Baeyer−Villiger Oxidation of cyclic ketones is well-docu-
mented in the literature.
Oxidative lactonization of diols represents an alternative7
route to lactones, but, though long-known, is far less explored.
Recently, we have applied the oxidative lactonization of 1,4-
butanediol as a “smart cosubstrate approach” to render alcohol
dehydrogenase (ADH)-catalyzed Meerwein−Ponndorf−Verley
As shown in Table 1, 1,4- and 1,5-diols were converted
smoothly by the HLADH−LMS system, whereas ε-caprolac-
tone (5b) formation (from 1,6-hexanediol, 5a) proceeded
rather sluggishly, reaching only 26% conversion even after
prolonged reaction times. The latter result is in apparent
contrast to the spectrophotometrically determined activity of
HLADH toward 1,6-hexanediol (see Supporting Information,
Figure S2). However, it should be kept in mind here that this
assay presumably only covers the first step of the lactonization
cascade (i.e., HLADH-catalyzed formation of the aldehyde). It
remains to be elucidated whether the slow accumulation of ε-
caprolactone is due to poor activity of HLADH toward the 7-
membered lactol, or if the lactol formation itself is rate-limiting.
NMR analysis of the reaction mixtures, however, revealed no
detectable aldehyde accumulation but indicated lactol accumu-
lation at roughly the same level as the lactone product. At the
same time, significant acidification of the reaction mixture was
8
reductions irreversible. Among other ADHs, horse-liver
alcohol dehydrogenase (HLADH) excelled by its high activity
toward 1,4-butanediol. Therefore, we became interested in
further exploring the scope of the HLADH-catalyzed oxidative
lactonization of diols. Particularly, the oxidative lactonization of
some meso- and chiral diols was investigated with respect to the
formation of enantiomerically enriched lactones. Furthermore,
we aimed at raising the reactant concentrations to preparatively
more relevant levels.
For regeneration of the oxidized nicotinamide cofactor
+
(
NAD ) we chose the recently established laccase−mediator
system (LMS) utilizing the robust laccase from Myceliophthora
thermophila (Mtlaccase) and acetosyringone as redox media-
tor. Overall, an aerobic chemoenzymatic cascade for the
9
Received: July 9, 2013
oxidative lactonization of diols as outlined in Scheme 1 was
envisioned.
Revised: September 17, 2013
Published: September 20, 2013
©
2013 American Chemical Society
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dx.doi.org/10.1021/cs400535c | ACS Catal. 2013, 3, 2436−2439

ACS Catalysis
Letter
Scheme 1. Oxidative Lactonization of Diols to the Corresponding Lactones Catalyzed by Horse Liver Alcohol Dehydrogenase
a
(HLADH)
a
+
Aerobic regeneration of the oxidized nicotinamide cofactor (NAD ) is achieved by using the so-called laccase-mediator system (LMS) comprising
the laccase from Myceliopthora thermophila (Mtlaccase) and acetosyringone as redox mediator (the structure of acetosyringone and the proposed
NADH-oxidation mechanism is shown in the Supporting Information, Figure S10). n = 1−3.
a
Table 1. HLADH−LMS-Mediated Oxidative Lactonization Reactions
a
+
−1
Reaction conditions: c(diol) = 50 mM, c(NAD ) = 0.5 mM, c(HLADH) = 0.3 g L , c(Mtlaccase) = 2 μM, c(acetosyringone) = 0.2 mM, buffer:
b
Tris-HCl (50 mM, pH 8), T = 30 °C, reaction time: 24 h. Determined spectrophotometrically, see Supporting Information for full experimental
details and results. Determined after 24 h.
c
observed which may explain the rather poor product yield.
Further investigations clarifying these issues are currently
underway.
group as efficiently as in β-position leading to a poor kinetic
resolution of the racemic 2a (vide infra).
In contrast to the poor kinetic resolution of 2a, the oxidative
desymmetrization of 3-methyl-1,5-pentanediol (4a) proceeded
smoothly with exquisite stereoselectivity (S), giving access to an
interesting building block in the total synthesis of, for example,
The oxidation reactions proceeded with high chemo- and
regioselectivity. Occasionally, traces of the putative lactol
intermediates were observable via GC/MS analysis, but all
disappeared in the course of the reactions. No indications for
the formation of dialdehydes (as dead-end products) could be
found. Also, in case of 1,4-pentanediol (2a), no indications for
the oxidation of the secondary OH-group (and formation of the
hydroxyketone) could be found. Unfortunately, the enantio-
meric purity of the corresponding lactone product (2b) was
very low (maximally 17% ee) and decreased with increasing
conversion of the starting material. Possibly, HLADH does not
discriminate chiral centers in γ-position to the reacting OH-
(
R,Z)-5-muscenone, tulearin C, 7,20-diisocyanoadociane, neo-
10
peltolide macrolactone, and (S)-methanophenazine.
Encouraged by these results, we evaluated the possibility of
increasing the substrate concentration to more preparatively
interesting values. Hence, we performed the oxidative
lactonization of 250 mM 1,4-butanediol while keeping all
other parameters the same (Table 1). It is worth mentioning
here that for these experiments efficient supply of O was
2
essential (data not shown), which becomes clear considering
the low solubility of O in aqueous media (around 0.25 mM).
2
2
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ACS Catalysis
Letter
Nevertheless, also under optimized O -intake (after increasing
the liquid−gas surface area by performing the reaction in 5-fold
enlarged reaction vessels) accumulation of γ-butyrolactone
under the conditions of a 2LPS setup, and full conversion could
not be reached. In fact, acetosyringone significantly partitioned
into the organic phase (K = 0.68, corresponding to >85%
extraction into the organic phase under the reaction
conditions) and thereby became unavailable for the regener-
ation system, whereas the dianion ABTS should not suffer from
such limitations. Indeed, using ABTS as mediator (at 1 mM
levels instead of 0.2 mM, to compensate for the lower activity
of the ABTS-based LMS) 95% conversion of 1a could be
reached within 24 h (Figure 1), thereby doubling the
2
(
1b) always ceased at approximately 120 mM (48%
conversion) and then gradually decreased over time, accom-
panied by a significant acidification of the reaction mixture to
approximately pH 5 (see Supporting Information, Figure S1 for
further details). The suspected hydrolysis of γ-butyrolactone
could be confirmed by GC/MS analysis (see Supporting
Information, Table S6). In fact, this hydrolysis was also
observed with the other lactone products evaluated (Table 1).
This spontaneous hydrolysis was undesired for various reasons.
First, it diminished the yield of the desired lactone.
Furthermore, the associated acidification of the reaction
mixture led to decreased HLADH-activity (see Supporting
Information, Figure S3) and impaired the stability of the
11
nicotinamide cofactor. Also, a slight product inhibition was
observed for HLADH in the presence of lactones (see
Supporting Information, Figure S4) requiring attention.
To circumvent these limitations, we envisioned a two-liquid-
12
phase system (2LPS) approach for the in situ extraction of the
less hydrophilic lactone products into an organic phase, thereby
circumventing or at least alleviating the lactone hydrolysis issue
(
Scheme 2).
Figure 1. HLADH-catalyzed oxidation of diols to their corresponding
Scheme 2. Using a Two-Liquid-Phase Approach to Alleviate
Lactone Hydrolysis
lactones using the 2LPS approach (1b(▼), 2b( ), 3b( ), 4b( ),
■
●
▲
a
5b(⧫)). Reaction conditions (aqueous, 0.2 mL): c(diol) = 250 mM,
+
c(NAD ) = 0.5 mM, c(HLADH) = c(Mtlaccase) = 2 μM, c(ABTS) = 1
mM, organic phase: DIPE (containing 5 mM dodecane, 1.8 mL),
Vorganic/Vaqueous = 9, buffer: Tris-HCl (50 mM, pH 8), T = 30 °C, 600
rpm. Yields are average values of duplicates.
conversion as compared to acetosyringone (see Supporting
Information, Figure S5 for the comparison). As shown in
Figure 1, this approach was successfully applied to the oxidative
lactonization of diols 1a−5a.
Gratifyingly, the enantioselectivity of the lactonization of 4a
was not impaired under these conditions. Again the optical
purity of 2b was not constant during the reaction as it
decreased from 17% after 1.5 h (first sample taken) to near-
racemic after 24 h (see Supporting Information, Figure S9,
Table S4).
Admittedly, further improvements are necessary to turn the
proposed 2LPS approach for oxidative lactonization of diols
into a truly practical option for the synthesis of (optically pure)
lactones. Especially, the identification of solvents for the
selective extraction of the lactone products will be the focus of
further investigations. As mentioned above, oxidative lactoniza-
7
tion of diols has been known since the 1970s. However, since
a
The lactone formed is partially extracted into the organic layer
the pioneering works by Jones et al. the substrate loadings have
not been improved significantly (10−25 mM). Possibly,
spontaneous lactone hydrolysis prevented higher concentra-
tions. With the current proof of concept, we have demonstrated
a promising solution to the lactone hydrolysis issue. Further
improvements (especially optimized organic phases with better
partitioning coefficients for the lactone products) will result in
practical systems for (chiral) lactone synthesis.
thereby avoiding its hydrolysis. n = 1−3.
As the presence of organic solvents may impair the
13
biocatalysts’ stability, we focused on solvents reported to
14
have negligible influence on HLADH stability. Though ethyl
acetate exhibited good extraction properties of the lactones (see
Supporting Information, Table S2), we decided against it as
hydrolysis (this time of the solvent) might lead to undesired
acidification. Therefore, for the proof-of-concept experiments,
we used diisopropyl ether (DIPE) as organic phase. In this
setup acetosyringone was substituted by 2,2′-azino-bis(3-
ethylbenzthiazoline-5-sulfonic acid (ABTS). The reason was
that the performance of the overall system significantly dropped
ASSOCIATED CONTENT
■
*
S
Supporting Information
Further details are given in Figures S1−S10 and Tables S1−S6.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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ACS Catalysis
AUTHOR INFORMATION
Letter
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E-mail: f.hollmann@tudelft.nl. Fax: +31 (0)15 2781415.
*
(
Notes
1
(
2
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Deutsche Bundesstiftung Umwelt (DBU)
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