Unveiling the Hidden Performance of Whole Cells in the Asymmetric Bioreduction of Aryl-containing Ketones in Aqueous Deep Eutectic Solvents

Article abstract of DOI:10.1002/adsc.201601064

In this contribution, we report the first successful baker's yeast reduction of arylpropanones using deep eutectic solvents (DESs) as biodegradable and non-hazardous co-solvents. The nature of DES [e.g. choline chloride/glycerol (2:1)] and the percentage of water in the mixture proved to be critical for both the reversal of selectivity and to achieve high enantioselectivity on going from pure water (up to 98:2 er in favour of the S-enantiomer) to DES/aqueous mixtures (up to 98:2 er in favour of the R-enantiomer). As a result, both enantiomers of valuable chiral alcohols of pharmaceutical interest were prepared from the same biocatalyst by simply switching the solvent. The possible inhibition of some (S)-oxidoreductases making part of the genome of such a wild-type whole cell biocatalyst when DESs are used as co-solvents may pave the way for an anti-Prelog reduction. The scope and limitations of this kind of biotransformations for a range of aryl-containing ketones are also discussed. (Figure presented.).

Full text of DOI:10.1002/adsc.201601064

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DOI: 10.1002/adsc.201601064
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Unveiling the Hidden Performance of Whole Cells in the
Asymmetric Bioreduction of Aryl-containing Ketones in Aqueous
Deep Eutectic Solvents
Paola Vitale,a,* Vincenzo Mirco Abbinante,a Filippo Maria Perna,a
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Antonio Salomone,b Cosimo Cardellicchio,c and Vito Capriatia,*
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`
Dipartimento di Farmacia–Scienze del Farmaco, Universita degli Studi di Bari “Aldo Moro”, Consorzio C.I.N.M.P.I.S.,
Via E. Orabona, 4, I-70125 Bari, Italy
Fax: (+39) 080 5442539; phone: (+39) 080 5442174; e-mail: paola.vitale@uniba.it; vito.capriati@uniba.it
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universitꢀ del Salento, Prov.le Lecce-Monteroni, I-73100
Lecce, Italy
b
c
CNR-ICCOM, Dipartimento di Chimica, Via E. Orabona 4, I-70125 Bari, Italy
Received: September 25, 2016; Revised: December 3, 2016; Published online: && &&, 0000
21 Dedicated to Professor Herbert Mayr on the occasion of his retirement
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Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201601064
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considering that more than half of the drug molecules
currently in use are chiral and contain at least one
stereogenic center.[1] Despite the tremendous advan-
ces in AC, however, developments of environmentally
friendly synthetic protocols especially in chiral drug
product manufacturing represent a challenge, and
toxic and hazardous volatile organic solvents are still
the solvents of choice in medicinal chemistry. En-
zyme-based biocatalysis represents a formidable tool
for asymmetric green chemistry development. Thus,
not surprisingly, it has received a great deal of
attention for the industrial production of biologically
active molecules.[2]
Wild-type whole-cell biocatalysts are often chosen
as biocatalysts because they are cheaper than isolated
and purified enzymes,[3] easy to handle, and come with
a continuous source of enzymes and efficient internal
cofactor regeneration systems [e.g. NAD(P)H]. Their
disadvantages are sometimes represented by lower
yields and/or selectivities because of competing reac-
tions catalyzed by other enzymes present in the whole
cells (e.g. hydrolysis processes), the low solubility of
organic reactants in water, low substrate loadings, and
low volumetric and catalyst productivities. To over-
come these drawbacks, many research groups have
focused their attention on the discovery of new strain
microorganisms (wild-type or recombinants), and on
the metabolic and evolutionary engineering of known
Abstract: In this contribution, we report the first
successful baker’s yeast reduction of arylpropa-
nones using deep eutectic solvents (DESs) as
biodegradable and non-hazardous co-solvents. The
nature of DES [e.g. choline chloride/glycerol (2:1)]
and the percentage of water in the mixture proved
to be critical for both the reversal of selectivity and
to achieve high enantioselectivity on going from
pure water (up to 98:2 er in favour of the S-
enantiomer) to DES/aqueous mixtures (up to 98:2
er in favour of the R-enantiomer). As a result, both
enantiomers of valuable chiral alcohols of pharma-
ceutical interest were prepared from the same
biocatalyst by simply switching the solvent. The
possible inhibition of some (S)-oxidoreductases
making part of the genome of such a wild-type
whole cell biocatalyst when DESs are used as co-
solvents may pave the way for an anti-Prelog
reduction. The scope and limitations of this kind of
biotransformations for a range of aryl-containing
ketones are also discussed.
Keywords: Deep
Eutectic
Solvents;
Green
chemistry; Whole-cell catalysis; Ketones; Reduc-
tion; Alcohols
54 Asymmetric catalysis (AC) is a type of catalysis in biocatalysts.[4] After the pioneering work by Zaks and
55 which a chiral catalyst addresses the formation of a Klibanov in 1984 describing enzymatic catalysis in
56 particular stereoisomer of a chiral compound. This is conventional organic media,[5] biocatalysis in uncon-
57 in particular central to the pharmaceutical industry ventional, nonaqueous reaction media sparked a surge
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of interest among practitioners, particularly after the octanol by the biocatalytic reduction of 2-octanone
introduction of second-generation ionic liquids (ILs) with Acetobacter pasteurianum GIM1.158 cells[10f] and
derived from readily available, renewable natural the asymmetric oxidation of 1-(4-methoxyphenyl)
products, and of so-called deep eutectic solvents ethanol with Acetobacter sp. CCTCC M209061
(DESs).[6]
The latter are the result of the correct combination
cells.[10g,h]
Genomes of wild-type whole cells often comprise a
of two or three safe and inexpensive high-melting- complex mixture of enzymes with different, sometimes
point hydrogen bond donors (HBDs) and acceptors opposite, enantioselectivities. Whole cells of baker’s
able to undergo self-association so as to achieve a yeast have also been traditionally used in preparative/
10 significant depression of the freezing point. DESs are industrial scale conditions for the enantioselective
11 generally obtained by mixing and gently warming a reduction of prochiral ketones to enantiomerically
12 quaternary ammonium halide salt [e.g. choline enriched chiral secondary alcohols, usually with S-
13 chloride (ChCl)] with metal salts or a HBD [e.g. urea, stereo-preference (Prelog’s rule), the latter being
14 glycerol (Gly)], but a combination of a carbohydrate, valuable synthons for the synthesis of chemicals,
15 a urea derivative, and an ammonium salt in varying pharmaceuticals, and flavours.[11] We questioned
16 ratios is also known. Thanks to their shallow ecolog- whether the innate disadvantages of whole cells such
17 ical footprint, attractive low prices, non-flammability, as baker’s yeast (vide supra) could be turned into an
18 biodegradability, ease of preparation with no further inherent advantage offering the possibility of synthe-
19 purification, low volatility, low toxicity, and recycla- sizing the opposite enantiomers of a chiral building
20 bility, DESs nowadays represent a nascent class of block simply starting from the same whole-cell bio-
21 sustainable solvents with ever-increasing applications catalyst. Inspired by the recent report by Maugeri and
22 in several fields spanning organic synthesis,[7a–i,o–q] Domınguez de Marıa on the use of different DES-
23 metal polishing,[7e] extraction and separation proces- aqueous mixtures to control the enantioselective
24 ses,[7j,k] polymerization and material sciences,[7l,m] and reduction of ethyl acetoacetate,[10a] (Scheme 1a) and
25 biomass processing.[7n] Applications of DESs in orga- building on our recent findings in using non-conven-
26 nocatalysis,[8a–d] organometallic chemistry,[8e–k] metal- tional yeasts in whole cell biocatalytic processes[12] and
27 catalyzed reactions,[8l–q] solar technology,[8r] and bio- DESs for exploring novel paradigms,[7o,8b,d,e,g,h,j,q] here-
28 transformations[8s,t] are still somewhat unexplored in we investigate baker’s yeast bioreduction of a range
29 fields, but with encouraging and particularly interest- of aryl-containing ketones using DESs as co-solvents
30 ing developments in the last few years. As per (Scheme 1b). The scope and limitation of this method-
31 biocatalytic reactions, not only have DES-aqueous- ology in terms of a successful tuning of stereo-
32 media mixtures been shown to be excellent solvents preference are also discussed.
33 for biotransformations catalyzed by a variety of
´
´
34 enzymes including lipases, epoxide hydrolases, pro-
35 teases, and peroxidases, but they have also signifi-
36 cantly contributed to enhancing the activity and
37 stability of these enzymes, and to facilitating high
38 substrate loadings, which is crucial for industrial
39 applications.[6,9] The use of DESs as effective reaction
40 media for whole-cell biocatalysis is still in its infancy,
41 and only a very few examples have been reported to
42 date. These include: the enantioselective reduction of
43 ethyl acetoacetate by whole cells of Saccharomyces
44 cerevisiae (baker’s yeast) in ChCl-based eutectic
45 mixtures with variable amounts of water,[10a,b] the
46 enantioselective reduction of a variety of aromatic
47 ketones in a DES (ChCl/Gly):buffer 80:20 v/v cata-
48 lyzed by whole cells of Escherichia coli overexpressing
49 specific oxidoreductases as biocatalysts,[10c] the dehy-
50 drogenation of cortisone acetate in ChCl/urea-water
Scheme 1. Enantioselective reduction reactions catalyzed by
bakerꢂs yeast in different DES–water mixtures.
51 mixtures catalyzed by immobilized whole cells of
52 Arthrobacter simplex,[10d] the asymmetric reduction of
53 3-chloropropiophenone with immobilized Acetobacter
54 sp. CCTCC M209061 cells in a ChCl/urea DES as the
55 most suitable co-solvent,[10e] and the combination of a
As a bench reaction, we set out to investigate the
56 biphasic system consisting of a DES and a water enantioselective reduction of phenylacetone 1a to 1-
57 immiscible IL for both the efficient synthesis of (R)-2- phenylpropan-2-ol 2a in DES systems catalyzed by
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Table 1. Stereoselective reduction of ketone 1a catalyzed by bakerꢂs yeast in different DES-aqueous systems[a]
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3
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Entry
Solvent
Time
[d]
Yield
(%)[b]
Abs.
er[d]
config.[c]
1
2
3
4
5
6
water
1
5
5
5
5
6
88
0
31
20
14
88
S
–
S
R
R
S
98:2
–
89:11
67:33
80:20
94:6
9
DES A[e]
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
DES A+40 w% water[e]
DES A+20 w% water[e]
DES A+10 w% water[e]
DES B+50
w% water[f]
7
8
9
DES B+40
6
6
6
53
44
36
R
R
R
52:48
80:20
98:2
w% water[f]
DES B+20
w% water[f]
DES B+10
w% water[f]
10
11
DES B[f]
6
6
0
5
–
S
–
DES C+10
81:19
w% water [g]
DES D+10
12
6
–
–
–
w% water [h]
[a] Reaction conditions: ketone 1a (1.5 mM), bakerꢂs yeast (230 mg mLÀ1) at 378C.
[b] Calculated by H NMR of the crude reaction mixture; no other products were detected.
[c] Absolute configuration of the major enantiomer.
[d] Enantiomeric ratio (er) determined by HPLC.
[e] DES A: ChCl/D-fructose (3:2 w/w) (50 g).
1
[f] DES B: ChCl/Gly (1:2 mol/mol) (50 g).
[g] DES C: ChCl/D-glucose (2:1 mol/mol) (50 g).
[h] DES D: ChCl/urea (1:2 mol/mol) (50 g).
36 one of the multiple alcohol dehydrogenases (ADHs) marxianus CBS 6556 growing cells.[12a] The replace-
37 present in the cheap and commercially available ment of water by neat ChCl/D-fructose DES mixture
38 baker’s yeast (Table 1). Alcohol 2a is a crucial synthon (3:2 w/w) did not lead to any conversion of 1a to 2a
39 in the preparation of L-deprenyl, a neuro-protective for up to 5 days (Table 1, entry 2), whereas the
40 drug currently used in the treatment of neurodegener- employment of a ChCl/D-fructose eutectic mixture
41 ative disorders such as Alzheimer’s and Parkinson’s with 40 w% water provided 2a with an er as high as
42 disease,[13] as well as amphetamine and its analogues, 89:11, the yield being 31% after 5 days incubation at
43 which are known to be potent central nervous system 378C (Table 1, entry 3).
44 stimulants used in the treatment of attention deficit
These results are consistent with the fact that a
certain amount of water is an absolute necessity for
45 hyperactivity disorder, narcolepsy, and obesity.[14]
46
A preliminary reaction run in tap water was done baker’s yeast whole cells in order for them to display
47 for comparison. After incubating a mixture of ketone their catalytic activity which is, however, also pre-
48 1a, baker’s yeast, and water (see Supporting Informa- served at long reaction times. On reducing the amount
49 tion) at 378C for 24 h, and centrifuging the slurry to of water to 20w%, the yield dropped to 20% but,
50 get the supernatant, extraction with EtOAc was interestingly, an inversion of stereoselectivity was
51 followed by purification of the crude by column observed this time in favour of the R-enantiomer (er=
52 chromatography on silica gel, furnishing the expected 67:33) (Table 1, entry 4). Finally, alcohol (R)-2a could
53 alcohol 2a in 88% yield and with an enantiomeric be recovered (14% yield) with an er 80:20 using DES
54 ratio (er) of 98:2 in favour of the S-enantiomer as a co-solvent with the addition of 10 w% water only
55 (Table 1, entry 1). Under these conditions, the stereo- (Table 1, entry 5). Since the nature of DES is known
56 selectivity gained proved to be higher than that to affect the cells’ viability and metabolism, whose
57 observed in the reduction of 1a by Kluyveromyces variation, in turn, influences both the activity and
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selectivity of the enzymes involved, as well as the transesterification and asymmetric carboligation proc-
regeneration of the coenzyme present in the whole esses.[17d–j]
cells,[10b] we also focused on different DES systems in
ChCl-based DESs used as solvents or co-solvents
order to even further improve the R-enantioselectivity. in aqueous solution have recently been shown to be
Satisfyingly, a second set of experiments performed in effective in enhancing the activity and stability of
the prototypical ChCl/Gly eutectic mixture (1:2 mol/ Candida rugosa lipase,[9e] horseradish peroxidase,[9f]
mol) and in the presence of different amounts of water and Penicillium expansum lipase,[9g] the properties as a
revealed a similar trend. As can be seen from the whole of the extensive H-bonding network typical of
results reported in Table 1, after 6 days incubation at DES mixtures most probably being the key to under-
10 378C, and by reducing the percentage of water from standing the beneficial effects of these neoteric
11 50 w% to 10 w%, the enantioselectivity shifted from solvents on enzyme activity. On the other hand, no
12 an er of 94:6 for the S-enantiomer of 2a (88% yield) certain interpretations have been made regarding the
13 to an er of 98:2 in favour of (R)-2a (36% yield) influence of DESs on the catalytic performance of
14 (Table 1, entries 6–9). An almost racemic mixture was whole cells, and a multitude of factors (e.g. protein
15 obtained with 40 w% water (Table 1, entry 7), where- denaturation, modification of cell membranes, enzyme
16 as, again, no bioconversion occurred in the absence of inhibition, etc.) may be playing a role.[10f] As transpires
17 water even after 6 days incubation at 378C (Table 1, from the data reported in Table 1, different results are
18 entry 10). Two additional ChCl-based DESs were also obtained simply by changing the nature of DES
19 investigated: ChCl/D-glucose (2:1 mol/mol) and ChCl/ components. Thus, while many isolated enzymes were
20 urea (1:2 mol/mol), each one with 10 w% water. In the shown to retain their activity in urea-based DESs,[6,9]
21 former case, under the best experimental conditions the presence of such a strong hydrogen-bond donor in
22 previously set up, the conversion dropped drastically an eutectic mixture exerted detrimental effect on
23 (up to 5%) and 2a was recovered with an er of 81:19 baker’s yeast catalysis (Table 1, entry 12). To the best
24 but in favour of the S-enantiomer (Table 1, entry 11). of our knowledge, the baker’s yeast-promoted reversal
25 Therefore, the inversion of enantioselectivity did not of enantioselectivity in DES-aqueous mixtures has
26 take place. On the other hand, upon switching to only been reported in the bioreduction of prochiral b-
27 ChCl/urea with 10 w% water no enzymatic activity ketoesters.[10a,b] One of the possible explanations
28 was noticed after 6 days incubation at 378C (Table 1, advanced for this phenomen involves the enantiose-
29 entry 12). Two aspects of these results are worth lective inhibition of some ADHs with S-stereoprefer-
30 noting. The first is that both the opposite stereo- ence present in baker’s yeast, whereas ADHs with R-
31 selective bioreductions of phenylacetone (1a) can be selectivity remain active.[10a]
32 successfully carried out using the inexpensive and
From this perspective, the bioreduction of prochi-
33 commercially available baker’s yeast as a wild-type ral ketone 1a was further investigated by evaluating
34 whole-cell biocatalyst. The second noteworthy aspect the kinetics of yeast ADHs both in water and in a
35 is that an important chiral building block such as 1- ChCl/Gly-based (DES B) mixture with 10 w% water
36 phenyl-2-propanol (2a) can be obtained with opposite at different reaction times. In pure water, the expected
37 and high enantioselectivity simply on switching from S-configured alcohol 2a was isolated after only 4 h in
38 water to an appropriate DES/water-based mixture as a 51% yield with an er value of 98:2, which suffers no
39 non-hazardous, unconventional medium.
erosion up to 48 h. After 72 h incubation in water,
40
To date, the chiral inversion of alcohol (S)-1a to however, a slight erosion in the er of (S)-2a was
41 (R)-1a, en route to both enantiomers of amphetamine, detected: 91:8 (yield: 88%) (Figure 1a; see also
42 has been achieved by exploiting a two-sequence path- Supporting Information). On the other hand, in the
43 way including a nucleophilic substitution with a presence of the above DES-water mixture, it took up
44 mixture of Et3N/AcOH followed by hydrolysis of the to 72 h to detect (R)-2a in the crude in 7% yield (1H
45 corresponding ester, although with partial racemiza- NMR analysis) but enantiomerically enriched up to
46 tion.[15]
47
96:4 er. After 120 h, the baker’s yeast-catalyzed
In general, in AC and when enantio-control is bioreduction of 1a in DES-water as a medium
48 dominated by central chirality, a reversal in the afforded (R)-2a in 36% yield and with an er value of
49 stereoselectivity takes place by changing the config- 98:2. (Figure 1b; see also Supporting Information).
50 uration of the stereocenter(s) of the chiral catalyst.[16] These observations strongly suggest the following: (a)
51 Up to now, strategies for enhancing the enantioselec- bioreduction catalyzed by S-ADHs (normally overex-
52 tivity of biocatalytic systems have made use of pressed in baker’s yeast) takes place at a higher
53 medium-engineering tactics such as the modification reaction rate in pure water and with no competition
54 of substrates[17a–c] and/or the use of organic additives with the reaction catalyzed by ADHs with R-stereo-
55 (e.g. esters, chiral amines, organic solvents, ILs) preference up to 48 h; (b) ChCl/Gly eutectic mixture
56 mainly applied in the bioreduction of b-ketoesters, in acts as an efficient inhibitor of S-ADHs, thereby
57 the kinetic resolution and hydrolysis of esters, and in paving the way for catalysis promoted by pro R-
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Figure 1. Time course of the biocatalytic reduction of ketone 1a by bakerꢂs yeast at 378C in water (a) and in a ChCl-Gly-
based mixture (DES B) with 10 w% water (b). (Each value reported represents the mean of three experiments. One
experiment is shown as an example. Deviation of repetitions never exceeded 10%. The experimental error associated with 1H
NMR and HPLC measurements has been calculated to be within the range of 2–3% and 0.5–1%, respectively).
23 ADHs, which is, however, effective only at much (1b) provided, after 3 days incubation at 378C in
24 longer reaction times. The low conversion rates water with baker’s yeast, the corresponding chiral
25 observed may be related either to the low concen- alcohol (S)-2b in 82% yield and with an er of up to
26 trations of R-ADHs in the whole cells of Saccharo- 97:3 er (Table 2, entry 1). When this substrate was
27 myces cerevisiae or to their low affinity towards the incubated in DES B with 20 w% water, an inversion
28 substrate. Catalytic activity of R-oxidoreductases, of absolute configuration for the major enantiomer
29 however, competitively affected the final enantiomeric again took place, and alcohol (R)-2b was formed with
30 enrichment of (S)-2a also in pure water starting from an er 91:9 in 14% yield (Table 2, entry 2). By reducing
31 72 h. Similarly, the enantioselective reduction of ethyl the amount of water to 10 w%, the er value increased
32 acetoacetate to the corresponding ethyl (R)-3-hydrox- to up to 95:5, the conversion being 10% (Table 2,
33 ybutanoate catalyzed by baker’s yeast in mixtures of entry 3). The 3-(trifluomethyl)phenylacetone (1c),
34 water with the ChCl/Gly DES, was observed only for which has a strong electron-withdrawing group on the
35 long reaction times (>200 h).[10a] The role played by phenyl ring, was reduced to (S)-2c by baker’s yeast in
36 water in tuning the conversion and enantioselectivity water both in high yield (90%) and high er (99:1),
37 is intriguing. Recent investigations established that the whereas the enantiomer (R)-2c could be recovered
38 dilution of DESs in water results in a progressive from DES B with 10 w% with an er of 70:30 and in
39 rupture of the hydrogen-bond network between the 15% yield (Table 2, entries 4,5). On the other hand, in
40 starting material. The supermolecular structure of the presence of a strong-electron-donating group on
41 DESs, however, is still preserved up to 50% water the phenyl ring (1d), reduction was seriously ham-
42 dilution. Further dilution produces an aqueous solu- pered, and the corresponding alcohol (2d) did not
43 tion containing the “free” forms of the solvated form in either the water or the eutectic mixture
44 individual components.[18] In this respect, the switch in (Table 2, entries 6,7). The replacement of the terminal
45 stereoselectivity sometimes observed in the biocata- methyl group in 1a by CF3 (compound 1e) gave an
46 lytic reduction of prochiral ketones may be due to a almost racemic mixture of alcohol 2e in water (49:51)
47 subtle interplay of “solvation” of whole cells and the whose er, however, incresed to up to 62:38 (12% yield)
48 selective “inhibition” of some enzymes according to again in favour of the R-enantiomer after 6 days
49 the percentage of water in the mixture, with the more incubation at 378C in DES B with 10 w% (Table 2,
50 diluted solution exhibiting physico-chemical proper- entries 8,9).
51 ties quite close to that of pure water.
We next evaluated the effect on the enantioselec-
52
The biocatalytic anti-Prelog reduction of phenyl- tivity when the spacer length between the carbonyl
53 acetone (1a) to (R)-1-phenyl-2-propanol (2a) success- moiety and the phenyl group was extended to two
54 fully conducted in DES-water mixtures led us to atoms. 1-Phenoxy-2-propanone (1f) successfully
55 examine the scope and limitations of this kind of underwent reduction by baker’s yeast in water deliver-
56 transformation in order to prepare other optically ing optically active (S)-1-phenoxy-2-propanol (2f) in
57 active secondary alcohols. (4-Fluorophenyl)acetone 89% yield and 91:9 er after 24 h incubation at 37 8C
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Table 2. Stereoselective reduction of ketones 1b–h catalyzed by bakerꢂs yeast in water and in a ChCl/Gly-aqueous system[a]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Entry
Compound
Solvent
Time [d]
Product [yield %][b]
Abs. config.[c]
er[d]
1
2
3
4
5
6
7
8
1b
1b
1b
1c
1c
1d
1d
1e
1e
1f
water
3
5
5
1
6
1
6
1
6
1
5
1
6
6
7
1
6
2b (82)
2b (14)
2b (10)
2c (90)
2c (15)
2d[f]
S
R
R
S
R
–
97:3
91:9
95:5
99:1
70:30
–
DES B+20 w% water[e]
DES B+10 w% water[e]
water
DES B+10 w% water[e]
water
DES B+10 w% water[e]
water
2d[g]
–
–
2e (>95)
2e (12)
2f (89)
2f (33)
2g (50)
2g (38)
2h (31)
2h[g]
S
R
S
S
S
S
S
–
49:51[h]
62:38[h]
91:9
88:12
91:9
62:38
83:17
–
9
DES B+10 w% water[e]
water
10
11
12
13
14
15
16
17
1f
DES B+10 w% water[e]
water
1g
1g
1h
1h
1i
DES B+10 w% water[e]
water
DES B+50 w% water[e]
water
2i[g]
–
–
–
–
1i
DES B+10 w% water[e]
2i[g]
[a] Reaction conditions: ketone 1b–i (1.5 mM), bakerꢂs yeast (200 mg mLÀ1) at 378C.
[b] Calculated by H NMR of the crude reaction mixture; no other products were detected.
1
[c] Absolute configuration of the major enantiomer.
[d] Enantiomeric ratio (er) determined by HPLC.
38 [e] DES B: ChCl/Gly (1:2 mol/mol) (50 g).
[f] Not determined because of the trace content.
39
40
41
[g] No reaction.
[h] Er determined by GC.
42
43
44 (Table 2, entry 10). Moving on DES B with 10 w% complete racemization when performed by Kluyver-
45 water as the reaction medium, the enantioselectivity omyces marxianus CBS 6556 yeast growing cells (96 h
46 dropped to 88:12 but still in favour of the S- incubation in water at 308C).[12a] Therefore, the longer
47 enantiomer, after 5 days incubation at 378C with a the spacer between the aromatic portion and the
48 yield of 33% (Table 2, entry 11). Similarly, baker’s carbonyl group, the lower the S-selectivity in the
49 yeast-catalyzed reduction of 4-phenyl-2-butanone (1g) reduction of prochiral ketones. Similarly, using re-
50 provided optically active (S)-4-phenyl-2-butanol (2g) combinant short-chain RasADH from Ralstonia sp.
51 in 50% yield and 91:9 er after 24 h incubation in DSM 6428 overexpressed in Escherichia coli, the
52 water, and in 38% yield and 62:38 er after 6 days stereoselective reduction of small-bulky ketones
53 incubation at 378C in DES B with 10 w% water proved to be highly substrate-dependent with some-
54 (Table 2, entries 12,13). Hence, a switch in stereo- what contrasting results in terms of stereoselectivity.[19]
55 preference was not observed in the case of ketones 1f
As per aromatic ketones, the baker’s yeast-medi-
56 and 1g. It is worth noting that the bioreduction of 1g ated acetophenone (1 h) reduction was relatively low
57 to 2g was proven to proceed in 74% yield, but with also in water providing the corresponding alcohol 2 h
Adv. Synth. Catal. 2016, 358, 1–10
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in only 31% yield after 6 days incubation at 378C and tionalized with strong electron-withdrawing groups
in 83:17 er according to the Prelog’s rule (Table 2, (e.g. CF3) either in the aryl moiety or at the position
entry 14). Conversely, on replacing the water with a next to the carbonyl group. On the other hand, by
ChCl/GlyÀwater mixture, the bioreduction of 1 h did increasing the spacer length between the aryl and the
not occur at all even in the presence of 50 w% water carbonyl groups to two atoms a change in the stereo-
and after 7 days incubation at 378C (Table 2, en- preference did not take place in eutectic mixtures, and
try 15). In general, the bioreduction of aromatic aromatic and heteroaromatic ketones could not even
ketones is notoriously more difficult with conventional be reduced. Up to now, the baker’s yeast-catalyzed
yeasts, and a higher stereoselectivity has only been bioreduction with reversal of stereoselectivity in
10 observed when using recombinant whole cells over- unconventional media has only been reported in the
11 expressing oxidoreductases as biocatalysts even in case of b-ketoesters.[10a,b] Thus, these results enlarge
12 unconventional media.[10c,19] A lower enantioselectivity even further the horizons of whole-cell biocatalysis in
13 (74:26 er, S:R) was instead achieved with Kluyveromy- these neoteric mixtures. Although under optimized
14 ces marxianus CBS 6556 growing cells.[12a] Finally, conditions for the enantioselective anti-Prelog reduc-
15 upon switching to an heteroaromatic ketone such as tion of arylpropanones R-ADHs showed a narrow
16 1i, which has a 2-furanyl moiety, baker’s yeast substrate pattern, physico-chemical properties of DES
17 reduction to the corresponding secondary alcohol 2i mixtures can be fine-tuned by the correct selection of
18 took place neither in water not in the eutectic mixture specific partners within certain chemical classes. Thus,
19 (Table 2, entries 16,17).
by using enzymes of cheap and commercially available
20
In summary, we first reported that baker’s yeast whole cells in novel biodegradable and environmen-
21 exhibits a fascinating switch in the rate of reaction and tally friendly DESs, not only may their notoriously
22 enantioselectivity in the reduction of arylpropanones hidden performance be revealed and exploited, but it
23 by simply changing the solvent from water to DES- is very promising and of great interest for developing
24 water mixtures, which is not a trivial matter. In a sustainable biocatalytic chemistry, thereby boosting
25 particular, the R-selectivity was surprisingly high (er its application in industry.[21] Further investigations
26 up to 98:2), even if at rather long reaction times (up to into related processes for other substrates using
27 6 days), when a ChCl/Gly (1:2) eutectic mixture was custom-tailored DES mixtures are ongoing in our
28 used and the reaction medium, in addition, contained laboratories.
29 up to 10 w% water. The obtained chiral secondary
30 alcohols serve as precursors for valuable products;
31 e.g., enantioenriched (S)- and (R)-1-phenylpropan-2- Experimental Section
32 ol (2a), which are key synthons for the preparation of
General Procedure
33 neuroprotective drugs. Our results are consistent with
34 a selective inhibition of S-oxidoreductases in aqueous Baker’s yeast bioreduction in ChCl/Gly (1:2 mol/mol,
35 ChCl/Gly eutectic mixtures, which paves the way for DES B)Àwater mixtures: DES B was prepared by
36 an R-ADH-mediated bioreduction catalyzed by en- gently heating and stirring at 608C for 5 min the
37 zymes present at low concentrations.
corresponding individual components (28.5 g of Gly
38
The final outcome in DES mixtures, however, has and 21.5 g of ChCl kept in an Erlenmeyer flask) until
39 also proven to depend on the nature of DES a clear solution was obtained. Then water [5 mL (10%
40 components. Thus, the replacement of D-fructose by w/w)] was added to the DES kept at 378C, and the
41 D-glucose did not similarly produce a change in baker’s yeast[22] (12.5 g) was dispersed to give a
42 stereochemistry, the resulting metabolic microenviron- smooth paste in the mixture. Ketone (1a–g) (100 mg)
43 ment, most probably, being as a whole still quite was added, and the mixture was stirred at 378C in an
44 similar to that of water. Glucose-based DESs are orbital shaker (250 rpm), monitoring the reaction’s
45 known to act as solvents and substrates when isolated progress by 1H NMR. After a fixed time (Table 1), the
46 enzymes are used.[20] On the other hand, upon switch- reaction was stopped by the addition of EtOAc
47 ing to ChCl/urea with 10 w% water, a complete knock followed by centrifugation, decantation, and extrac-
48 out of whole-cell biocatalyst took place as no tion with EtOAc. The extracts were dried over
49 enzymatic activity was detected. Thus, the final stereo- anhydrous Na2SO4, and the solvent evaporated under
50 selectivity achieved may be the result of a subtle reduced pressure. The residue was purified by silica
51 interplay of “solvation” of whole cells and the gel column chromatography using hexane/EtOAc
52 selective “inhibition” of some enzymes according to (90:10 or 60:40) as an eluent to yield the desired
53 the nature of DES components and the percentage of alcohol (2a–g). Spectral data are in agreement with
54 water in the mixture.
those previously reported (see Supporting Informa-
55
Chiral enantioenriched R-configured alcohols can tion).
56 still be obtained in aqueous ChCl/Gly eutectic mix-
57 tures starting from arylpropanone derivatives func-
Adv. Synth. Catal. 2016, 358, 1–10
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Chem. 2013, 125, 3152–3163; Angew. Chem. Int. Ed.
2013, 52, 3074–3085; e) E. L. Smith, A. P. Abbott, K. S.
Ryder, Chem. Rev. 2014, 114, 11060–11082; f) J. Garcꢃa-
ꢇlvarez, In Handbook of Solvents, Vol. 2, 2nd Edn:
Use, Health, and Environment ed. (Ed.: G. Wypych),
ChemTec Publishing, Toronto, 2014; g) A. Paiva, R.
Craveiro, I. Aroso, M. Martins, R. L. Reis, A. R. C.
Duarte, ACS Sustain. Chem. Eng. 2014, 2, 1063–1071;
h) P. Liu, J.-W. Hao, L.-P. Mo, Z.-H. Zhang, RSC Adv.
2015, 5, 48675–48704; i) D. A. Alonso, A. Baeza, R.
Chinchilla, G. Guillena, I. M. Pastor, D. J. Ramꢈn, Eur.
J. Org. Chem. 2016, 612–632; j) F. Pena-Pereira, J.
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Acknowledgements
This work was financially supported by the University of Bari
within the framework of the Project “Sviluppo di nuove
metodologie di sintesi mediante l’impiego di biocatalizzatori e
solventi
a
basso impatto ambientale” (code: Per-
na01333214Ricat), and the Interuniversities Consortium
C.I.N.M.P.I.S. The authors are also indebted to Prof.
Gennaro Agrimi for valuable discussions, and to Mr Roberto
Capobianco for his contribution to the Experimental Section.
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Unveiling the Hidden Performance of Whole Cells in the
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