Productivity enhancement of CC bioreductions by coupling the in situ substrate feeding product removal technology with isolated enzymes

Article abstract of DOI:10.1039/c1cc16014a

To overcome the usually low productivities of the CC bond bioreduction of α,β-unsaturated aldehydes we combined the in situ substrate feeding product removal (SFPR) technology with a cascade system comprising an isolated ene-reductase and a chemoselective alcohol dehydrogenase.

Full text of DOI:10.1039/c1cc16014a

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COMMUNICATION
Q
Productivity enhancement of C C bioreductions by coupling the in situ
substrate feeding product removal technology with isolated enzymesw
Elisabetta Brenna,a Francesco G. Gatti,*a Daniela Monti,*b Fabio Parmeggiania and
Alessandro Sacchetti*a
Received 27th September 2011, Accepted 20th October 2011
DOI: 10.1039/c1cc16014a
Q
To overcome the usually low productivities of the C C bond
bioreduction of a,b-unsaturated aldehydes we combined the
in situ substrate feeding product removal (SFPR) technology
with a cascade system comprising an isolated ene-reductase and
a chemoselective alcohol dehydrogenase.
The baker’s yeast (b.y.) mediated reduction of activated
alkenes1 suffers from several drawbacks: low substrate loading,
non-quantitative conversion, long reaction time, troublesome
work-up, and competitive side reactions, that overall contribute
to give modest productivities (o0.5 g LÀ1
d
À1).2 So far, two
strategies have been adopted to address some of these issues.
The first one relies on the in situ substrate feeding product
removal (SFPR) technology,3 which ensures, by adsorption of
substrate and products on a hydrophobic resin, very low concen-
trations, preventing toxic effects. However, despite its application
leading to an improvement of yield, work-up, and in certain cases
of enantioselectivity,3b the productivities are still unsatisfactory.
Alternatively, the second strategy is based on isolated
ene-reductases (ERs, e.g. the Old Yellow Enzymes OYE2–OYE3
of b.y.)4 which often allowed us to obtain quantitative conversions
without side-products.5 Nevertheless, the productivities are usually
too low, since high substrate concentrations cannot generally
be employed (5–10 mM). Also the aqueous–organic biphasic
systems have been tested for the reduction of a set of
a-methylcinnamaldehyde derivatives.5e However, the effects were
contrasting, indeed, the ee of products improved with respect to
those achieved in the homogeneous phase, whereas the yields
resulted worse. This example reveals another critical aspect of
using the ERs for the reduction of a-substitued aldehydes since
the products racemise in water even at almost neutral pH.
Thus, we envisioned that the goal of a substantial increase in
productivity might be pursued by the combination of these
Scheme 1 Enantioselective biocatalysed reductions of aldehydes 1a–4a.
two complementary strategies. For this purpose we compared
their applications (either separated or combined) to the reduction
of a set of a-substituted aldehydes (1a–4a, Scheme 1), which are
for several reasons of some interest in our ongoing research
program.
The b.y.-mediated reduction of 1a gave alcohol 1c (precursor
of the antidiabetic Navaglitazar) in a modest yield of 36% and
with a low ee of 55% (Table 1, entry 1). An improvement has
been accomplished by applying the SFPR strategy (entry 2),1c
however the productivity (0.39 g LÀ1 À1) remained way too
d
low. The use of isolated OYEs in homogeneous phase, together
with a glucose dehydrogenase (GDH from Bacillus megaterium)6
for NADPH cofactor regeneration, gave quantitative conver-
sions, but disappointingly the ees of saturated aldehyde 1b
resulted lower (90% and 86% ee with OYE2 and OYE3,
respectively, entries 3 and 4) than those previously obtained.
Instead, the SFPR technology combined with isolated OYEs,
under conditions comparable to those used with whole cells,
afforded 1b with a good ee of 96% and in a 65% yield, which
allowed a 10-fold increase of productivity (entry 5). To further
optimise the system, we assessed the effect of different resin/
substrate ratios (Xr/s) and substrate loadings on enzyme
performance (entries 6–9). It is noteworthy that high Xr/s
values allow us to obtain 1b with a high ee but a lower
conversion, whereas quantitative yields could be achieved with
a Politecnico di Milano, Department of Chemistry, Materials and
Chemical Engineering ‘‘Giulio Natta’’, Piazza Leonardo Da Vinci
32, 20131, Milano, Italy. E-mail: francesco.gatti@polimi.it,
alessandro.sacchetti@polimi.it; Tel: +39 02 23993070
b Istituto di Chimica del Riconoscimento Molecolare, C.N.R.,
Via Mario Bianco, 9, 20131, Milano, Italy.
E-mail: daniela.monti@icrm.cnr.it; Tel: +39 02 28500038
w Electronic supplementary information (ESI) available: Experimental
methods, NMR spectra of products 1b, 2b, 3b and 4c, and representative
chiral GC or HPLC chromatograms. See DOI: 10.1039/c1cc16014a
c
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Chem. Commun., 2012, 48, 79–81 79

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Table 1 Baker’s yeast and ene-reductases mediated reductions of a,b-unsaturated aldehydes 1a–4a
Entry Substrate
Biocatalyst
Product Loading/g LÀ1 Xr/s/g gÀ1 Time/d Yielda [%] eeb [%] Productivity/g LÀ1 dÀ1
1
2
3
4
5
6
7
8
9
10
b.y.
b.y.c
1c
1c
1b
1b
1b
1b
1b
1b
1b
1b
1
2
1
—
10
—
—
10
3
1
3
1
—
4
4
36
81
100
100
65
92
100
60
55
95
90
86
96
94
95
93
94
n.d.
0.07
0.39
1.90
1.86
19.11
26.77
29.10
34.74
47.72
—
OYE2
OYE3
OYE2
OYE2
OYE2
OYE2
OYE2
OYE2d
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
15
15
15
30
30
15
82
22
11
12
13
14
b.y.
2c
2b
2b
2b
1
1
1
6
20
—
—
1
4
38
92
0.08
—
—
e
OYE2
OYE3
OYE2
0.5
0.5
0.5
—
—
n.d.
n.d.
90
e
97
11.06
15
OYE3
2b
6
1
0.5
94
90
10.72
16
17
18
19
20
21
b.y.
3c
3b
3b
3b
3b
1
1
1
10
10
10
20
—
—
3
1
1
4
20
81
—
29
86
94
60
65
n.d.
83
73
99
0.04
1.34
—
5.31
14.88
18.71
OYE2
OYE3
OYE2
OYE2
0.5
0.5
0.5
0.5
0.5
OYE2 + ADH 3c
22
23
24
25
26
27
28
29
30
31
32
33
b.y.
b.y.
b.y.
4c
4c
4c
4b
4b
4b
4b
4b
4b
1
1
1
1
1
10
10
10
10
1
10
15
20
—
—
1
3
1
1
—
1
4
4
4
36
29
23
85
87
81
58
8
60
85
99
7
11
50
69
84
80
95
99
95
0.07
0.07
0.06
0.91
OYE2
OYE3
OYE2
OYE2
OYE2
OYE3
OYE2 + ADH 4c
OYE2 + ADH 4c
OYE3 + ADH 4c
0.5
0.5
0.5
0.5
0.08
0.08
0.5
0.5
0.5
0.97
12.15
9.80
9.20
10.13
1.19
9
61
88
76
10
10
17.42
14.82
1
Standard expt. cond.: (i) for b.y.: 0.45 g mLÀ1 wet cells, 90 mg mLÀ1 glucose, tap water, 30 1C; (ii) for OYE: 150 mg mLÀ1 OYE, 4 U mLÀ1 GDH, 0.1 mM
NADP+, 4 eq. glucose, 50 mM phosphate buffer, pH 7.0, 30 1C, 160 rpm; (iii) for OYE + ADH: the same as (ii), 2 U mLÀ1 HLADH, 0.1 mM
b
c
d
e
NAD+.a Determined by GC or after standard work-up. Determined by chiral HPLC or GC. Ref. 1c. Ref. 7. The starting material decomposed.
lower Xr/s at the expense of a slight decrease of optical purity.
Under optimised conditions, an improved productivity of
29.1 g LÀ1 dÀ1 was reached while maintaining a still good ee
of 95% and a quantitative yield (entry 7). The crucial role of
the SFPR approach was further emphasised by repeating the
latter reaction with the same substrate loading, but in the
absence of the resin and employing the portion-wise addition
protocol.7 Indeed, with substrate 1a the yield decreased to an
unacceptable 22% (entry 10). These results suggested the
following conclusions: (i) the saturation concentrations of
1a and/or 1b somehow inhibit the enzymatic activity of ERs;
(ii) the racemisation of 1b occurs during the biotransformation;
(iii) the latter is slower than the b.y. mediated reduction of the
carbonyl group to give the more stable alcohol 1c; and (iv) in the
presence of the resin the optical purity of aldehyde 1b is
preserved, since the racemisation is partially or completely
suppressed. To verify the occurrence of racemisation, a sample
of (S)-1b left under the homogeneous phase biotransformation
conditions, but without any enzyme, showed an appreciable loss
of optical purity after the same reaction time. In contrast, the
same sample preserved its ee when it was adsorbed on the resin.
The reduction of aldehyde 2a illustrates another important
feature of our methodology. In this case the reduction in
homogeneous phase failed, since the starting material decomposed
before being converted into the saturated aldehyde 2b
(entries 12 and 13).8 Again, the combination of ERs with the SFPR
technology led to a considerable improvement of productivity
(two orders of magnitude) without any decomposition. More-
over, no appreciable loss of optical purity was observed, since
the enantioselectivity achieved with both enzymes (ee 90%,
entries 14 and 15) resulted comparable to that attained with
b.y. (92% ee, entry 11).
Especially interesting is the reduction of aldehyde 3a to give
either the alcohol 3c or the saturated aldehyde 3b, precursors
of Rotigotine,9 a drug used for the treatment of Parkinson’s
disease. This substrate, in which the CQC double bond is
inserted into a six-membered ring, is much more sterically
hindered than 1a and 2a. Indeed, its reduction with b.y. gave 3c
in a lower yield (20%) and with a modest ee of 60% (entry 16).
Unexpectedly, a very different substrate specificity was found,
since the OYE3-catalysed reduction failed, whereas OYE2
gave 3b in a yield of 81% and with a slight enhancement
of ee (entry 17). Even in this case the application of the
combined strategy allowed a substantial improvement affording
3b with a better optical purity (73% ee) and productivity (entry
20 vs. entry 16).
c
80 Chem. Commun., 2012, 48, 79–81
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racemic 4b gave 4c without appreciable optical enrichment,
we ruled out any DKR.
Finally, the synergic combination of this cascade system
(OYE, ADH, GDH) together with the SFPR applied to 3a
allowed us to obtain 3c with a 99% ee and a 470-fold
productivity enhancement compared to b.y. (entry 21 vs. 16).
In conclusion, we have shown that the isolated enzymes
(such as ERs, ADHs and GDH) are compatible13 with the
in situ SFPR technology. This methodology allows a substantial
enhancement of productivity, opening a new perspective on
the exploitation of recombinant ERs in preparative organic
synthesis. Moreover, we found that the use of resin might be
vital for the reduction of potentially unstable substrates,
preventing either starting materials decomposition or products
racemisation. In particular, the latter can be further minimised
by coupling an ADH in cascade to an ER in such a way to
reduce chemoselectively the unstable a-substitued aldehyde, as
soon as it is formed, to the more robust alcohol.
Scheme 2 Cascade synthesis of 4c by coupling of an OYE with
HLADH.
Finally, despite its structural similarity to 3a, very different
results were obtained in the case of 4a, a possible intermediate in
the synthesis of the antidepressant Robalzotan.9 The reduction
was first studied with b.y. and SFPR (entries 22–24). By increasing
the Xr/s value the optical purity of alcohol 4c improved up to
99% ee, but at the expense of a lower yield (entry 24), since
both substrate and product concentrations in water are lower,
and consequentially the conversion rate and the racemisation
rate decrease. The reduction with isolated ERs in homogeneous
phase (entries 25 and 26) showed that a fast racemisation of
aldehyde 4b occurred, since the latter was recovered almost
racemic. Disappointingly, even our methodology turned out to
be insufficient to suppress such a racemisation (entries 27 and 28).
In an attempt to improve the optical purity we also tested a much
shorter reaction time that provided better ees but too low
conversions (entries 29 and 30).
Notes and references
1 (a) R. Csuk and B. I. Glanzer, Chem. Rev., 1991, 91, 49;
¨
(b) S. Serra, C. Fuganti and F. G. Gatti, Eur. J. Org. Chem.,
2008, 1031; (c) E. Brenna, C. Fuganti, F. G. Gatti and
F. Parmeggiani, Tetrahedron: Asymmetry, 2009, 20, 2694;
(d) E. Brenna, C. Fuganti, F. G. Gatti, L. Malpezzi and S. Serra,
Tetrahedron: Asymmetry, 2008, 19, 800.
2 Productivity = [Loading  Yield/100  (ee + 100)/200]/Time.
3 (a) J. T. Vicenzi, M. J. Zmijewski, M. R. Reinhard, B. E. Landen,
W. L. Muth and P. G. Marler, Enzyme Microb. Technol., 1997,
20, 494; (b) P. D’Arrigo, C. Fuganti, G. Pedrocchi Fantoni and
S. Servi, Tetrahedron, 1998, 54, 15017; (c) I. Hilker,
Thus, we wondered whether the co-presence of alcohol
dehydrogenases (ADHs) with ERs might play a determinant
role in the achievement of higher ees of 4c. A screening of
several commercially available ADHs allowed us to select the
horse liver alcohol dehydrogenase (HLADH) as the most
promising enzyme to be coupled with isolated ERs (data not
shown). This cascade system (OYE2–3, HLADH, GDH,
NADPH and NADH) was tested in homogeneous phase or
coupled to the SFPR technology (Scheme 2). In the best case
(OYE2, HLADH, SFPR, entry 32), as soon as the saturated
aldehyde 4b was formed, the HLADH reduced chemoselectively
the latter over the unsaturated aldehyde 4a to give the alcohol 4c
with an excellent ee of 99%. The productivity was improved as
well to 17.4 g LÀ1 dÀ1, a 250-fold increase with respect to that
achieved with yeast. Although a plethora of ADHs have been
isolated and successfully employed for the reduction of many
carbonyl compounds,10 such a chemoselectivity has never been
reported; furthermore, very recently, an ADH has been combined
with an ER in a telescopic sequence (one pot two steps in series)
without taking advantage of such a selectivity,11 which might
allow the set-up of a more appealing cascade system.
M. C. Gutierrez, V. Alphand, R. Wohlgemuth and R. Furstoss,
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Org. Lett., 2004, 6, 1955; (d) I. Hilker, M. C. Gutierrez,
R. Furstoss, J. Ward, R. Wohlgemuth and V. Alphand, Nat.
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4 (a) K. Stott, K. Saito, D. J. Thiele and V. Massey, J. Biol. Chem.,
1993, 268, 6097–6106; (b) H. S. Toogood, J. M. Gardiner and
N. S. Scrutton, ChemCatChem, 2010, 2, 892–914.
5 (a) C. Stueckler, M. Hall, H. Ehammer, E. Pointner, W. Kroutil,
P. Macheroux and K. Faber, Org. Lett., 2007, 9, 5409; (b) M. Hall,
C. Stueckler, B. Hauer, R. Stuermer, T. Friederich, M. Breuer,
W. Kroutil and K. Faber, Eur. J. Org. Chem., 2008, 1511;
(c) S. K. Padhi, D. J. Bougioukou and J. D. Stewart, J. Am. Chem.
Soc., 2009, 131, 3271; (d) J. F. Chaparro-Riggers, T. A. Rogers,
E. Vazquez-Figueroa, K. M. Polizzi and A. S. Bommarius, Adv. Synth.
Catal., 2007, 349, 1521; (e) C. Stueckler, N. J. Mueller, C. K. Winkler,
S. M. Glueck, K. Gruber, G. Steinkeller and K. Faber, Dalton Trans.,
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8 A sample of 2a (pH 7.0, 30 1C, 1% DMSO) completely decomposed
after 12 hours.
9 Rotigotine and Robalzotan synthesis will be reported elsewhere.
10 M. M. Musa and R. S. Phillips, Catal. Sci. Technol., 2011, 1, 1311.
11 M. Korpak and J. Pietruszka, Adv. Synth. Catal., 2011, 353, 1420.
12 D. Giacomini, P. Galletti, A. Quintavilla, G. Gucciardo and
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13 In a previous report the use of SFPR with isolated enzymes caused
a loss of catalytic activity: F. Zambianchi, S. Raimondi, P. Pasta,
G. Carrea, G. N. Caggero and J. M. Woodley, J. Mol. Catal. B:
Enzym., 2004, 31, 165.
In addition, since it is known that HLADH promotes the
dynamic kinetic resolution (DKR) of a-arylpropionaldehydes
in phosphate buffer (pH 7.4), to give the corresponding
alcohols with good ees,12 we were concerned whether the high
optical purity of 4c could be ascribed also to the HLADH or
exclusively to the OYEs. Since the HLADH reduction of
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 79–81 81