Enantioselective preparation of δ-valerolactones with horse liver alcohol dehydrogenase

Article abstract of DOI:10.1002/cctc.201300640

Horse liver alcohol dehydrogenase (HLADH) has been found to be a versatile biocatalyst for the desymmetrization of prochiral 3-arylpentane-1,5-diols, based on a two-step one-pot oxidation. This procedure has allowed the formation of valuable (S)-lactones in good to excellent conversions and enantiomeric excess. The catalytic performance of HLADH has been studied using several cofactor regeneration systems and cosolvents, finding great improvements in terms of activity with L-lactate dehydrogenase, while the stereoselectivity of the process was significantly improved when using tetrahydrofuran. Docking studies has revealed the pattern substitution importance in the selectivity and activity of this oxidative process. Not just horsing around: Horse liver alcohol dehydrogenase is found to be a versatile biocatalyst for the desymmetrization of 3-arylpentane-1,5-diols through a two-step one-pot oxidation. The catalytic performance of horse liver alcohol dehydrogenase (HLADH) is studied with several cofactor regeneration systems and cosolvents. Docking studies reveal the importance of pattern substitution in the selectivity and activity of this biotransformation.

Full text of DOI:10.1002/cctc.201300640

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DOI: 10.1002/cctc.201300640
Enantioselective Preparation of d-Valerolactones with
Horse Liver Alcohol Dehydrogenase
Alba Dꢀaz-Rodrꢀguez,[a] Javier Iglesias-Fernꢁndez,[b] Carme Rovira,[b, c] and Vicente Gotor-
Fernꢁndez*[a]
Horse liver alcohol dehydrogenase (HLADH) has been found to
be a versatile biocatalyst for the desymmetrization of prochiral
3-arylpentane-1,5-diols, based on a two-step one-pot oxida-
tion. This procedure has allowed the formation of valuable
(S)-lactones in good to excellent conversions and enantiomeric
excess. The catalytic performance of HLADH has been studied
using several cofactor regeneration systems and cosolvents,
finding great improvements in terms of activity with L-lactate
dehydrogenase, while the stereoselectivity of the process was
significantly improved when using tetrahydrofuran. Docking
studies has revealed the pattern substitution importance in
the selectivity and activity of this oxidative process.
only a few examples in the literature, in addition to early ex-
amples by Jones and co-workers, deal with the enzymatic de-
symmetrisation of prochiral 1,5-pentanediols through oxidative
processes.[4a,c,e]
Lactones are important compounds not only because of
their structural implications as basic chemicals but also be-
cause they are relevant building blocks found in natural prod-
ucts.[6] With the growing demand for enantiomerically pure
synthons, the biocatalysed preparation of chiral lactones re-
mains a challenging goal and different (chemo)enzymatic
routes have been applied by making use of lipase-mediated
hydrolysis of carboxylic acid derivatives,[7] Baeyer–Villiger bio-
oxidations of the corresponding cyclic ketones[8] or ADH-cata-
lysed bioreduction of ketones.[9]
Alcohol dehydrogenases (ADHs) are widely used to catalyse
the reduction and oxidation of carbonyl groups and alcohols,
respectively. These oxidoreductases have found important ap-
plications in both academia and industry for the production of
pharmaceuticals, fragrances and high value-added deriva-
tives.[1] Although the stereoselective bioreduction of prochiral
ketones is an attractive transformation for organic chemists,
the oxidation of alcohols is less appealing because in most of
the cases the chirality is lost. Thus, there are a limited number
of biocatalytic examples reporting on the oxidation of diols to
the corresponding lactones.[2] Remarkably, 1,4-butanediol has
recently been used as an irreversible cosubstrate for redox bio-
catalysis leading to g-butyrolactone, a thermodynamically
stable and kinetically inert co-product.[3] In this context, the
biocatalytic enantioselective oxidation of diols,[4] and in partic-
ular meso and prochiral diols, has been scarcely investigated.
Conceptually, this approach is a powerful strategy for the prep-
aration of optically active compounds because the substrate
loses one or more elements of symmetry.[5] To our knowledge,
Herein, we report an asymmetric approach for the synthesis
of enantioenriched lactones based on the desymmetrisation of
the corresponding 3-arylpentane-1,5-diols. The enantioselective
oxidation of prochiral diols for the preparation of these rele-
vant compounds with ADHs could be an alternative to the
commonly used protocols.
Table 1. Optimisation of the enzymatic reaction of 1a.
Entry[a]
pH
Cosolvent
c [%][b]
(S)-3a ee [%][c]
1[d]
2
3
4
5[e]
6[f]
7[g]
8
9
–
–
–
–
–
–
–
52 (1:1.8)
100 (1:16)
80 (1:4)
66 (1: 5.2)
70 (0:1)
46 (1:3)
34 (1:1)
84 (1:5.4)
72 (1:3)
61 (1:2)
48
49
50
38
44
50
45
54
64
74
50
60
7.5
6.5
9
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
[a] A. Dꢀaz-Rodrꢀguez, V. Gotor-Fernꢁndez
Department of Organic and Inorganic Chemistry
University of Oviedo
2% THF
5% THF
7% THF
2% CH3CN
7% CH3CN
C/Juliꢁn Claverꢀa, 33006 Oviedo (Spain)
Fax: (+34)985103448
E-mail: vicgotfer@uniovi.es
9
10
11
12
73 (1:3.2)
62 (1:2.6)
[b] J. Iglesias-Fernꢁndez, C. Rovira
Institut de Quꢀmica Teꢂrica i Computacional (IQTCUB)
Universitat de Barcelona
Martꢀ i Franquꢃs 1, 08028 Barcelona (Spain)
[a] Reactions conditions: 1a (25 mm), NAD+ (0.84 mm), HLADH (2 U) and
the recycling system in Tris buffer for 72 h at 308C unless noted other-
wise; [b] Conversion values were determined from GC analysis, consider-
ing the formation of hemiacetal 2a and lactone 3a, and their ratio is
given in parentheses; [c] ee values of 3a were determined from HPLC
analysis; [d] Flavin mononucleotide was used as a recycling system; the
reaction was performed at [e] 378C, [f] 208C and [g] 48C.
[c] C. Rovira
Instituciꢄ Catalana de Recerca i Estudis AvanÅats (ICREA)
Passeig Lluis Companys 23, 08020 Barcelona (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300640.
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In a first set of experiments, we screened a panel of ADHs in
the one-pot oxidation of the model substrate 3-phenylpen-
tane-1,5-diol (1a; Table 1) to prepare the corresponding enan-
tioenriched d-valerolactone 3a (Table 1). Of those ADHs, horse
liver alcohol dehydrogenase (HLADH) showed the highest ac-
tivity (see the Supporting Information for details) and therefore
it was selected for the optimisation of the biocatalytic process.
Formation of the valerolactone occurs by desymmetrization
of 1a, which is oxidized and generates an unstable hydroxy al-
dehyde that cyclises to afford the corresponding hemiacetal
(2a). The subsequent reoxidation of 2a gave access to the
stable lactone 3a.[10] On the basis of these initial results and to
use catalytic amounts of nicotinamide adenine dinucleotide
(NAD+) cofactor, we then attempted the enantioselective oxi-
dation of diol 1a via incubation with HLADH with flavin mono-
nucleotide as a recycling system.[4a] The reaction was moni-
tored by using GC, and a slow oxidation of diol 1a was ob-
served. Under the above-mentioned conditions, a mixture of
hemiacetal 2a and lactone 3a was observed in a 1:1.8 ratio,
which yielded the lactone 3a with a modest 48% ee after 72 h
(Table 1, entry 1). Notably, previously reported ee value for the
desymmetrisation of 1a was 21% ee.[4a] The biotransformation
proceeded with stereoselectivity towards the pro-S hydroxy
group, and preferentially, after reoxidation to the (S)-lactone.
To increase the conversion values towards the lactone, we
explored other recycling systems and reaction conditions. First-
ly, the addition of acetaldehyde to the reaction mixture was at-
tempted. Unfortunately, no reaction was observed, probably
owing to inhibition issues. On the basis of this unsuccessful
result with the coupled substrate approach, l-lactate dehydro-
genase (LDH) was used. This enzyme catalyses the reduction of
pyruvate to lactate with the concomitant oxidation of nicotin-
amide adenine dinucleotide to regenerate NAD+.[11] This regen-
eration system led to the total conversion of the process,
which yielded the (S)-lactone in moderate ee after 72 h
(Table 1, entry 2). The experimental results indicate that the
enantioselection in the 1a!2a step is the most important
contribution. The ee of the final lactone 3a remains unaltered
if the conversion values are between 50 and 100% (see the
Supporting Information for detailed time course study).
provement in the enantioselectivity was observed. The best re-
sults in terms of conversion and ee were obtained with THF
(Table 1, entries 8–10), and a 74% ee was observed in the pres-
ence of 7% of THF. However, the yield of 3a decreased consid-
erably in this case. Percentages of cosolvent higher than 15%
inhibited the oxidation reaction (data not shown). Solvent en-
gineering studies demonstrated that with acetonitrile as the
cosolvent, conversions were slightly lower than those obtained
with THF (Table 1, entries 11 and 12). Moreover, the addition of
a non-miscible cosolvent such as hexane enabled the addition
of higher percentages of cosolvent, which gave conversions
and ee values similar to those obtained in the aqueous
medium (data not shown).
Table 2. Effect of the substituent position and cosolvent on the enantio-
selective preparation of d-valerolactones 3a–g.
Entry[a]
R
THF [%]
c [%][b]
(S)-3a–g ee [%][c]
1
2
3
4
5
6
7
8
C6H5 (a)
C6H5 (a)
–
7
–
7
–
7
–
7
–
7
–
7
–
7
100 (1:9)
61 (1:2.5)
92 (1:17)
55 (1:3)
72 (1:2.6)
39 (2:1)
52
74
60
68
65
76
40
53
52
75
92
97
50
55
4-MeO-C6H4 (b)
4-MeO-C6H4 (b)
4-F-C6H4 (c)
4-F-C6H4 (c)
3-MeO-C6H4 (d)
3-MeO-C6H4 (d)
3-F-C6H4 (e)
3-F-C6H4 (e)
2-MeO-C6H4 (f)
2-MeO-C6H4 (f)
2-F-C6H4 (g)
2-F-C6H4 (g)
82 (1:9)
43 (1:1.8)
71 (1:3.5)
31 (1:0.8)
62 (1:15)
21 (1:3)
9
10
11
12
13
14
76 (1:12)
32 (1:2)
[a] Reaction conditions: substrates 1a–g (25 mm), NAD+ (0.84 mm), pyru-
vate (68 mm), LDH (2 U) and of HLADH (2 U) in Tris buffer (pH 7.5) at
308C for 72 h; [b] Conversion values were determined from GC analysis,
considering the formation of the corresponding hemiacetal and lactone,
and their ratio is given in parentheses; [c] The ee values of lactones were
determined from HPLC analysis.
At this point, different parameters were investigated to in-
crease the ee of this biotransformation. As expected, a change
in the pH of the reaction drastically affected the yield of the
process (Table 1, entries 3 and 4) without an increase in the ee
and a decrease in the 2a/3a ratio. Subsequently, the effect of
the temperature was studied, and although an increase in the
temperature favoured the formation of the thermodynamically
stable lactone 3a (notably, hemiacetal 2a was not detected at
378C; entry 5), the conversion of the process decreased drasti-
cally. Yet, if the reaction was performed at lower temperatures
(20 and 48C; entries 6 and 7, respectively), only lower conver-
sions were observed, indicating that both pH and temperature
could not be tuned to achieve better ee values.
We also extended this methodology to other 3-arylpentane-
1,5-diols 1b–g[13] (Table 2) to study the effect of the pattern
substitution in the aromatic ring on the lactonisation reaction.
From the results in Table 2, it becomes clear that the introduc-
tion of a group in the aromatic ring at the para position (1b
and c) led to a slight increase in the ee (Table 2, entries 3 and
5), which could owe to additional interactions in the active site
of HLADH. Nevertheless, the conversions for the oxidation of
these substrates decreased.
Motivated by the fact that oxidoreductases can work in or-
ganic solvents with changes in their properties,[12] a percentage
of cosolvent with respect to the buffer solution was added to
the reaction mixture (Table 1, entries 8–12). A significant im-
We then turned our attention to diols 1d–g possessing sub-
stituents closer to the prochiral centre (meta and ortho posi-
tions). We can observe that 3-(m-methoxyphenyl)pentanediol
(1d) reacts slower than the corresponding para-substituted
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diol (1b), and the ee is also lower (Table 2, entry 7). This result
suggests that the substrate binds less efficiently in the active
site. The same tendency was observed with 1e (Table 2,
entry 9). Moreover, when the methoxy group was placed at
the ortho position (1 f), it had a remarkable effect on the enan-
tioselectivity and a 92% ee (Table 2, entry 11) was observed. In
light of these results, we can hypothesise that the enantiose-
lectivity of the process is affected not only by the presence of
a substituent but also by its position, which may interact with
residues in the HLADH active site. However, for the substrate
1g containing a fluorine atom in the ortho position, the ee de-
creased again, probably owing to the small size of the sub-
stituent (Table 2, entry 13).
pare enantioenriched lactones, which expand the substrate
scope reported until now.[4a] Optical purities can be tuned by
adding organic cosolvents, in which THF has shown a beneficial
effect in terms of enantiodiscrimination and aqueous systems
lead to the isolation of the lactones in higher yields. The syn-
thesis of substituted diols has enabled the evaluation of a cer-
tain structure–activity relationship in the active site of HLADH.
We are currently investigating further opportunities in this
area, considering new target substrates and focusing on the in-
crease in the conversion values.
Experimental Section
To further rationalise the relation between the diol structure
and the enantioselectivity, docking studies were performed on
substrates 1a, g and f.[14] For the sake of simplicity, we focused
only on the desymmetrisation process. Although compounds
1a and g form an E–S complex in which the pro-R and pro-S
conformations resulted in the same structure, compound 1 f
demonstrates important differences in both conformers. An in-
tramolecular hydrogen bond interaction is observed for the
pro-S conformation (Figure 1 and see the Supporting Informa-
Enantioselective preparation of valerolactones 3a–g
General procedure: In a typical experiment, a diol 1a–g (25 mm)
was suspended in Tris buffer (50 mm, pH 7.5, 0.5 mL) and treated
with NAD+ (50 mL, 10 mm stock, 0.84 mm), sodium pyruvate (40 mL,
1m stock, 68 mm) and LDH (2 U). This mixture was shaken for
5 min, and finally, HLADH was added (2 U). The mixture was
shaken at 250 rpm in a rotatory shaker at 308C, extracted with
EtOAc (2ꢃ0.5 mL), dried over Na2SO4 and analysed by using GC to
determine the conversion value. HPLC analyses were performed to
determine the ee values. Control experiments in the absence of
enzyme were performed for all substrates, and the oxidation reac-
tion was not observed after long periods of time.
Acknowledgements
This research is part of the BIONEXGEN project (grant agreement
266025) sponsored by the European Union in the Seventh Frame-
work Programme (FP7 2007–2013).
Keywords: asymmetric synthesis · biocatalysis · enantiopure
lactones · molecular modelling · recycling system
Figure 1. Substrate 1 f in complex with HLADH. Left: Structural superposi-
tion of the precursors of the R (grey) and S (cyan) enantiomers; right: the
pro-R enantiomer is removed to show the intramolecular hydrogen bond in-
teraction of the pro-S enantiomer more clearly.
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