Asymmetric synthesis of (R)-3-hydroxy-2-methylpropanoate ('Roche Ester') and derivatives via biocatalytic C=C-bond reduction

Article abstract of DOI:10.1002/adsc.201000522

Enoate reductases from the 'old yellow enzyme' family were employed for the asymmetric bioreduction of methyl 2-hydroxymethylacrylate and its O-allyl, O-benzyl and O-TBDMS derivatives to furnish (R)-configurated methyl 3-hydroxy-2-methylpropionate products in up to >99% ee Variation of the O-protective group had little influence on the stereoselectivity, but a major impact on the reaction rate.

Full text of DOI:10.1002/adsc.201000522

UPDATES
DOI: 10.1002/adsc.201000522
Asymmetric Synthesis of (R)-3-Hydroxy-2-methylpropanoate
(ꢀRoche Esterꢁ) and Derivatives via Biocatalytic C=C-Bond
Reduction
Clemens Stueckler,a Christoph K. Winkler,a Melanie Bonnekessel,b
and Kurt Fabera,*
a
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28/2, 8010-Graz,
Austria
Fax : (+43)-316-380-9840; e-mail: Kurt.Faber@Uni-Graz.at
BASF SE, GVF/B-A030, 67056 Ludwigshafen, Germany
b
Received: July 2, 2010; Revised: September 4, 2010; Published online: October 7, 2010
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000522.
Abstract: Enoate reductases from the ꢀold yellow
enzymeꢁ family were employed for the asymmetric
bioreduction of methyl 2-hydroxymethylacrylate
and its O-allyl, O-benzyl and O-TBDMS derivatives
to furnish (R)-configurated methyl 3-hydroxy-2-
methylpropionate products in up to >99% ee Var-
iation of the O-protective group had little influence
on the stereoselectivity, but a major impact on the
reaction rate.
enones and nitroalkenes, whereas (di)carboxylic acids
and esters are transformed more slowly.
To illustrate the importance of this enzyme class for
asymmetric synthesis, we aimed at their applicability
for an industrially relevant product, that is, (R)-3-hy-
droxy-2-methylpropanoate, which is commonly denot-
ed as the ꢀRoche esterꢁ. The latter is a popular chiral
building block for the synthesis of vitamins (e.g., a-to-
copherol[6]), fragrance components (e.g., muscone[7]),
and antibiotics (e.g., calcimycin,[8] palinurin,[9] rapa-
mycin,[10] 13-deoxytedanolide,[11] dictyostatin[12]) and
natural products (e.g., spiculoic acid A[13]). Classical
methods for its preparation include the diastereose-
lective addition of non-racemic alcohols as chiral aux-
iliaries,[14] the transformation of a chiral homoallylic
acetate[15] or involve aldol condensation[16] and – most
prominent – the transition metal-catalysed asymmet-
Keywords: biocatalysis; C=C-bioreduction; enoate
reductase; old yellow enzyme; substrate engineer-
ing
The asymmetric reduction of C=C bonds creates (up ric hydrogenation of acrylate esters using Rh[17] (ee up
to) two chiral carbon centres and is thus one of the to 99%) or Ru[18] (ee up to 94%). For the biocatalytic
most widely employed strategies for the production of synthesis of the ꢀRoche esterꢁ only few examples are
chiral materials. The biocatalytic variant, which is ap- reported: the stereoselective oxidation of 2-methyl-
plicable to activated alkenes bearing an electron-with- 1,3-propanediol by Gluconobacter and Acetobacter
drawing substituent is catalysed by enoate reductases spp.[19] (ee up to 97%), the asymmetric reduction of
[EC 1.3.1.X],[1,2] which are members of the ꢀold yellow ethyl 4,4-dimethoxy-3-methylcrotonate using bakerꢁs
enzymeꢁ (OYE) family.[3] Over the past few years, in- yeast[20] and the stereoseletive (formal) b-hydroxyl-
creasing attention has been devoted to these flavo- ation of isobutyric acid using Pseudomonas putida
proteins[4] in view of their substrate scope,[5] encom- (ATCC 21244).[21] All of these biotransformations
passing a,b-unsaturated carbonyl compounds (such as were performed using whole (fermenting) microbial
enals and enones), as well as carboxylic acids and de- cells with several enzymes being involved. Only re-
rivatives thereof (such as esters, cyclic imides, nitriles, cently, was it shown that a non-flavin NADH-depen-
lactones) and nitroalkenes. As a rule of thumb, the dent D4,5-steroid 5b-reductase from Arabidopsis thali-
degree of activation of the C=C-bond exerted by the ana was able to reduce ethyl 2-hydroxymethylacry-
electron-withdrawing effect of the activating substitu- late, however, the stereochemistry of the product was
ent goes hand in hand with the substrate acceptance, not examined in regard to its absolute configuration
which ensures generally fast reaction rates for enals, and enantiomeric composition.[22]
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Clemens Stueckler et al.
UPDATES
methyl 3-hydroxy-2-methylpropionate (1b) by four of
the eleven enoate reductases tested (entries 1–8) with
perfect stereoselectivity (ee up to >99%), NCR,
NerA and OPT1 were inactive and XenA showed
limited stereoselectivites (eemax 63%, entry 15). De-
spite these encouraging selectivities, insufficient reac-
tion rates led to incomplete conversion of this sub-
strate (cmax 37%, entry 5). Since the primary allylic al-
cohol group of 1a is considerably hydrated in aqueous
solution, the corresponding allyl, benzyl and TBDMS
ethers were tested as more lipophilic substrate ana-
logues.
Scheme 1. Asymmetric bioreduction of methyl 2-hydroxy-
methylacrylate derivatives 1a–4a.
Overall, the masking of the hydroxy group of the
Herein we report on the synthesis of the ꢀRoche initial substrate by easily removable allyl and benzyl
esterꢁ via biocatalytic reduction of methyl 2-hydroxy- ether groups enhanced the substrate acceptance dras-
methylacrylate using eleven OYEꢁs. In order to test tically. The O-allyl analogue 2a and O-benzyl ether 3a
the influence of the overall substrate structure regard- were converted by OYEs1-3 and YqjM with approxi-
ing its size and polarity on the relative rate and/or ste- mately three-fold enhanced rates (cmax 90%, entry 5).
reoselectivity, various protective groups on the hy- In the case of NCR, NerA and OPR1 this substrate
droxy moiety were investigated.
modification even turned the inactive substrate 1a
Old yellow enzymes OYE1-3 from Saccharomyces into a suitable target molecule for these enzymes
sp.,[23] 12-oxophytodienoic acid reductase isoenzymes (substrate 3a, cmax 89%, entry 13), and the conversion
OPR1 and OPR3 from Lycopersicon esculentum,[24] of 2a using XenA was almost quantitative (entry 16).
YqjM from Bacillus subtilis,[25] nicotinamide-depen- In addition to improved reaction rates, the incomplete
dent cyclohexenone reductase (NCR) from Zymomo- stereoselectivities of YqjM and XenA were corrected
nas mobilis,[26] xenobiotic reductases A and B (XenA, from ees 94% and 60% to >99%, respectively (en-
XenB) from Pseudomonas putida and P. fluorescens, tries 8 and 16). However, the stereopreference re-
respectively,[27] glycerol trinitrate reductase (NerA) mained constant for all modified substrates by invari-
from Agrobacterium radiobacter[28] and estrogen-bind- ably yielding (R)-2b and (R)-3b.
ing protein (EBP1) from Candida albicans[29] were
tested for their ability to reduce four methyl 2-hy- sometimes may be controlled by the substrate shape,
droxymethacrylate derivatives (Scheme 1, Table 1).
such as (E/Z)-configuration of the C=C-bond,[4a,5c,e]
Since the stereopreference of enoate reductases
The unprotected Roche ester precursor methyl 2- we attempted to obtain the (S)-ꢀRoche esterꢁ by in-
hydroxymethylacrylate (1a) was reduced to (R)- creasing the steric bulkiness of the hydroxy protective
Table 1. Conversion and enantiomeric excess of bioreduction products 1b–4b.
Entry
Enzyme
Cofactor[a]
1b
ee [%]
2b
ee [%]
3b
ee [%]
4b
ee [%]
c [%]
c [%]
c [%]
c [%]
1
2
3
4
5
6
7
8
OYE1
OYE2
OYE3
YqjM
NCR
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
NADH
NAD+[b]
20
7
7
12
37
5
14
18
<1
n.d.
<1
n.d.
<1
n.d.
17
28
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
98 (R)
94 (R)
n.d.
n.d.
n.d.
n.d.
n.d.
14
5
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
18
3
14
7
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
<1
n.d.
<1
n.d.
<1
n.d.
6
20
14
24
<1
n.d.
5
26
65
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>99 (R)
>99 (R)
>99 (R)
>99 (R)
n.d.
38
10
90
32
26
69
34
22
16
30
77
76
84
97
66
17
67
55
55
20
39
35
89
84
76
90
9
10
11
12
13
14
15
16
NerA
OPR1
XenA
n.d.
>99 (R)
>99 (R)
>99 (R)
>99 (R)
n.d.
63 (R)
60 (R)
>99
[a]
Standard conditions: substrate 1a–4a (10 mM), NADH (15 mM), Tris-HCl-buffer 50 mM, pH 7.5, 308C, 24 h.
NAD+ (100 mM)/glucose dehydrogenase (10 U)/glucose (20 mM); c=conversion; n.d.=not determined.
[b]
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Asymmetric Synthesis of (R)-3-Hydroxy-2-methylpropanoate (ꢀRoche Esterꢁ) and Derivatives
group even further in order to enforce a flipped sub- Determination of Absolute Configurations
strate orientation within the active site of the enzyme.
Unfortunately, this attempt for selectivity control
The absolute configurations of 1b, 2b and 4b were deter-
mined by co-injection with reference material of known ab-
failed, as the bulky tert-butyldimethylsilyloxy deriva-
tive 4a invariably furnished (R)-4b in >99% ee, going
hand in hand with a negative effect on the reaction
rates: The activities dropped significantly (for YqjM,
NCR and OPR1) or were completely erased (for
OYE1-3 and NerA), only XenA was able to reduce
4a quantitatively with absolute selectivity for the (R)-
enantiomer (entry 16). None of the substrates could
be reduced with sufficient rates using OPR3, XenB
and EBP1 (c <2%).
In conclusion, an efficient method for the prepara-
tion of (R)-methyl 3-hydroxy-2-methylpropionate
[ꢀ(R)-Roche esterꢁ] and related O-protected deriva-
tives was developed via bioreduction of the corre-
sponding acrylate precursors was developed using
enoate reductases from the ꢀold yellow enzymeꢁ
family. In order to ensure full conversion, the allylic
hydroxy moiety had to be protected with an allyl or
benzyl ether group to render a more lipophilic sub-
strate analogue.
solute configuration. The absolute configuration of 3b was
determined by comparison with a published chiral HPLC
analysis (Chiralcel OD-H 0.46ꢃ25 cm, 98:2 heptane:2-prop-
anol mobile phase at 1.5 mLminÀ1 flow): the (R)-enantio-
mer eluted at 3.88 min, the (S)-enantiomer eluted at
4.17 min.[32]
Supporting Information
General information on commercially obtained compounds
and materials, the synthesis of substrates 1a–4a, the synthe-
sis of reference material for rac-1b, rac-3b and rac-4b, and
analytical methods for the determination of conversion and
enantiomeric excess are described in the Supporting Infor-
mation.
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