Oxidation-hydroxymethylation-reduction: A one-pot three-step biocatalytic synthesis of optically active α-aryl vicinal diols

Article abstract of DOI:10.1039/c1gc16092c

A one-pot multi-step approach comprising enzymatic oxidation- hydroxymethylation-reduction enables the synthesis of optically active α-aryl vicinal diols with high yields and enantioselectivities. Formaldehyde required for the hydroxymethylation step is enzymatically produced in situ using less hazardous methanol as substrate.

Full text of DOI:10.1039/c1gc16092c

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COMMUNICATION
Oxidation-hydroxymethylation-reduction: a one-pot three-step biocatalytic
synthesis of optically active a-aryl vicinal diols
Saravanakumar Shanmuganathan,a Dessy Natalia,a Lasse Greiner*a,b and Pablo Dom´ınguez de Mar´ıa*a
Received 1st September 2011, Accepted 15th November 2011
DOI: 10.1039/c1gc16092c
A one-pot multi-step approach comprising enzymatic
oxidation-hydroxymethylation-reduction enables the syn-
thesis of optically active a-aryl vicinal diols with high yields
and enantioselectivities. Formaldehyde required for the
hydroxymethylation step is enzymatically produced in situ
using less hazardous methanol as substrate.
by the capability of BAL to catalyze the hydroxymethylation
of aromatic aldehydes with formaldehyde to afford a-aryl-2-
oxo-1-hydroxy ketones.17–20 Alternatively, this step could also
be conducted via carbene-based organocatalysis,21–23 mimick-
ing the function of ThDP-lyases.24–26 Yet, in itself, the ef-
ficient organocatalytic approach requires either stoichiomet-
ric amounts of bases or elevated temperatures, thus com-
bining this with enzymatic steps in one-pot could prove
difficult.
In modern sustainable synthesis the required efficient and
selective protocols need to be aligned with minimal waste
production. Hazardous chemicals must be avoided or at least
efficiently converted to safer chemicals. To combine these
requirements with the supply of formaldehyde for the BAL-
catalyzed hydroxymethylation17,18 two strategies were devised:
A) Co-utilization of formaldehyde for cofactor regenera-
tion in the subsequent alcohol dehydrogenase (ADH) cat-
alyzed enantioselective ketone reduction (Scheme 1, route
A); or B) Enzymatic in situ formation of formaldehyde
through oxidation of the less hazardous methanol (Scheme 1,
route B).
The production of enantiopure building blocks for manufactur-
ing pharmaceuticals or biologically-active compounds usually
involves elaborate multi-step synthetic protocols. Typically,
isolation of intermediates and tedious protection-deprotection
steps are needed, often leading to low yields and high E-
factors. One powerful approach towards sustainable organic
synthesis lies in the development of one-pot multi-step syn-
theses, e.g. enzyme cascades inspired by the in vivo biosyn-
thesis. For the successful implementation of one-pot catalytic
multi-step reaction systems, stringent conditions need to be
orchestrated for optimal results. Reaction rates need balanc-
ing; compatibility of catalysts has to be achieved; and pro-
ductivity has to be optimized.1–3 A few multi-step catalytic
processes have been reported, comprising excellent examples
of enzymatic and metal-enzyme combinations.4–10 Furthermore,
recent elegant approaches combine organo- and biocatalysis as
well.11–12
As first step, the BAL-catalyzed carboligation of benzalde-
hyde and formaldehyde was carefully assessed to minimize the
excess of formaldehyde needed to allow quantitative conversion
in 1, as the competitive formation of benzoin potentially reduces
In this communication, we disclose
a one-pot multi-
enzymatic concept utilizing oxidoreductases and benzaldehyde
lyase (BAL), a thiamine-diphosphate-dependent (ThDP) lyase,
for the production of optically active a-aryl vicinal diols,
which are valuable building blocks for fine chemicals and
pharmaceuticals.13 The use of multi-step biocatalytic reactions
for the production of these important building blocks has
been testimonial, with only few examples involving lyases and
oxidoreductases (in two-pot reactions),14 or chemo-catalysis.15,16
Herein, the core step – a C–C bond formation – is enabled
aInstitut fu¨r Technische und Makromolekulare Chemie (ITMC), RWTH
Aachen University, Worringerweg 1, 52074, Aachen, Germany.
E-mail: dominguez@itmc.rwth-aachen.de; Fax: +49 241 8022177;
Tel: +49 241 8020468
bDECHEMA e.V. Karl-Winnacker-Institut, Theodor-Heuss-Allee 25,
D-60486, Frankfurt am Main, Germany. E-mail: greiner@dechema.de;
Fax: +49 697564388; Tel: +49 697564337
Scheme 1 Conceptual outline for the multi-step biocatalytic reactions.
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Scheme 2 BAL-catalyzed hydroxymethylation. 20 mL phosphate buffer 50 mmol L-1, pH 8.0, 5% v/v 2-MeTHF, 0.25 mmol benzaldehyde, 0.75 mmol
formaldehyde, 2.5 mmol L-1 MgSO4, 0.15 mmol L-1 ThDP, 10 U BAL.
Scheme 3 Enzymatic multi-step process for the enantioselective synthesis of (R)-2. 10 mL phosphate buffer 50 mmol L-1, pH 8.0, 5% v/v 2-MeTHF,
0.13 mmol benzaldehyde, 0.4 mmol formaldehyde, 2.5 mmol L-1 MgSO4, 0.15 mmol L-1 ThDP, 10 U BAL, 354 U GDH, 21 U FDH, NADH/NAD+
0.5 mmol L-1 each.
overall selectivity. 2-Methyltetrahydrofuran (2-MeTHF) was
employed as (bio-based) co-solvent, since it provides an excellent
operational window for BAL, while avoiding significant forma-
tion of wastewater and further environmental issues associated
with other co-solvents (e.g. DMSO, MTBE), leading to a
diminished E factor in the process.27 Thus, a 3-fold excess of
formaldehyde assured quantitative conversion in a-aryl-2-oxo-
1-hydroxy ketone 1 (Scheme 2).
Once the reaction conditions of the enzymatic hydroxymethy-
lation were optimized, the next step was to capitalize the excess
of formaldehyde (Scheme 2) as ancillary substrate (cofactor
regeneration) for the enantioselective ketone reduction. To this
end, glycerol dehydrogenase from Cellulomonas sp. (GDH) was
selected for the enantioselective reduction of 1.28 For cofac-
tor regeneration (NADH), formaldehyde dehydrogenase from
Pseudomonas putida was chosen.29 After careful optimization of
absolute and relative enzyme and cofactor loadings with reaction
times, high conversion and excellent enantioselectivity for the
desired product diol (R)-2 were achieved (isolated yield 75%, ee
> 99%) (Scheme 3).
Alternatively, to avoid issues related to toxicity inherent to
formaldehyde (still one equivalent present after hydroxymethy-
lation and oxidation steps), the enzymatic in situ formaldehyde
formation and consumption was assessed, thus providing the
basis for a “formaldehyde-free” hydroxymethylation. To this
end, the FAD-dependent alcohol oxidase from Hansenula sp.
proved to be an efficient catalyst for the oxidation of methanol
with oxygen.30,31 The coupled product H2O2 was converted by
catalase, regenerating oxygen in solution and avoiding detrimen-
tal effects to the enzymes. Firstly reactions were separated in
time, that is, enzymatic methanol oxidation being conducted
for 8 h, followed by BAL and benzaldehyde addition for
additional 16 h. 1 was isolated in ~93% yield along with
traces of benzoin. Presumably, enzymatic oxidation steps are
slower compared to the C–C ligation reaction. By systematically
varying enzyme loadings, yield of 1 was quantitative, and
remarkably colored impurities were diminished, compared to
route A (with addition of excess of formaldehyde). Finally, since
no deactivation of BAL in the presence of methanol or alcohol
oxidase was observed, oxidation and hydroxymethylation were
combined in one step. Gratifyingly, 1 was again formed in
quantitative conversion. Consequently, all three reactions were
conducted in a one-pot sequential array: first the in situ
oxidation of methanol to formaldehyde, followed by concomi-
tant BAL-catalyzed cross condensation with benzaldehyde, and
followed by Lb-ADH addition to afford (S)-2 quantitatively
(isolated yield) with ee > 99% (Scheme 4). It is important
to mention that in this case NADPH-dependent alcohol de-
hydrogenase from Lactobacillus brevis (LbADH) was used,32
affording the (S)-enantiomer. Thus, by smartly using different
enzymes of a toolbox, access to both enantiomers might be
achieved.
Finally, the methodology was extended to more challenging
aromatic aldehydes like furfural. Furan ethane diols can be
used as building blocks for the preparation of levoglucosenone,
a valuable synthon for biologically-sound intermediates.33–35
Traditional synthetic approaches involve Lewis acid-catalyzed
glucal rearrangement, typically with poor enantioselectivity.33–35
Since furfural-based diols are unstable, they were chemically
dibenzoylated in situ. The first approach following route A
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Scheme 4 Four enzymes in one-pot reaction: In situ oxidation-hydroxymethylation-reduction. 20 mL, phosphate buffer 50 mmol L-1, pH 8.0, 5%
v/v 2-MeTHF, 0.12 mmol benzaldehyde, 0.90 mmol methanol, 33 U alcohol oxidase, 14 U catalase, 2.5 mmol L-1 MgSO4, 0.15 mmol L-1 ThDP,
40 U BAL, 10 U LbADH. 0.5 mmol L-1 NADPH, 0.1 mmol L-1 FAD.
(Scheme 1) provided a successful proof-of-principle, with low
isolated yields (25%) albeit with excellent (R)-enantioselectivity
(Scheme 5).
loadings and less enzyme concentrations, eventual biocatalyst
recyclability, etc. In this respect, a promising approach for future
research may be the incorporation of whole-cells with overex-
pressed enzymes, as it has been successfully reported for lyases
or oxidoreductases in processes with enhanced enzyme stability
and higher substrate loadings.37,38 With such implementation
at hand, the smart combination of enzymatic cascade pro-
cesses together with high productivities and efficiencies may be
reached.
In summary one-pot multi-step biocatalytic concepts have
been reported, comprising oxidation, hydroxymethylation, and
a further reduction to afford chiral a-aryl vicinal diols. By
in situ production or its subsequent utilization as reducing
agent, the use of hazardous formaldehyde is addressed. Another
advantage is that the concept allows the use of different
oxidoreductases, providing access to both enantiomers (“on-
demand”) and furan derivatives. Future sustainable (indus-
trial) processes will adopt more and more such highly inte-
grated multi-catalytic steps with diminished waste formation,
together with optimized substrate concentration and enzyme
loadings.
With the considerable amount of 3 as intermediate despite the
higher amount of GDH applied, the bottleneck was supposed
to be at low activity of GDH towards furan compounds. In
fact, when LbADH was applied, with glucose dehydrogenase
(GlucDH) for cofactor regeneration, this resulted in excellent
isolated yields (87%) and enantioselectivity (>99% ee, (S))
(Scheme 6). Therefore, the herein reported conceptual approach
may accept other enzymes with different enantioselectivity or
different bias towards organic molecules. Due to the ample port-
folio of oxidoreductases already available,31,36 further successful
combinations may be expected.
By means of these strategies a significant reduction of the E
factor can be achieved, since the amount of organic solvent
(for product extraction), as well as water for the enzymatic
process can be reduced to one third, due to the setup of just one
final downstream processing. Importantly, these inherent envi-
ronmental advantages of the herein reported concept still need
to be complemented with optimized reaction times, substrate
Scheme 5 Biocatalytic formation of chiral furane-diols via route A. 40 mL phosphate buffer 50 mmol L-1, pH 8.0, 5% v/v 2-MeTHF, 1.6 mmol
furaldehyde, 4.8 mmol formaldehyde, 2.5 mmol L-1 MgSO4, 0.15 mmol L-1 ThDP, 168 U BAL, 1500 U GDH, 95 U FDH, NADH/NAD+ 1 mmol
L-1 each.
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Scheme 6 Biocatalytic formation of chiral furan-diols. 40 mL phosphate buffer 50 mM, pH 8.0, 5% v/v 2-MeTHF, 1.6 mmol furaldehyde, 3.6 mmol
glucose, 4.4 mmol CaCO3, 4.8 mmol formaldehyde, 2.5 mmol L-1 MgSO4, 0.15 mmol L-1 ThDP, 168 U BAL, 50 U Lb-ADH, 2800 U GlucDH,
0.5 mmol L-1 NADPH.
18 A. Cosp, C. Dresen, M. Pohl, L. Walter, C. Ro¨hr and M. Mu¨ller,
Acknowledgements
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Financial support from DFG training group 1166 “BioNoCo”
(“Biocatalysis in non-conventional Media”) is gratefully ac-
knowledged. Mrs. Hannelore Eschmann and Ms. Julia Wurlitzer
(both RWTH Aachen University) are acknowledged for support
in analytical measurements (HPLC).
22 R. Hackstell, I. Jovel, M. Beller, M. Hateley, C. Weckbecker and K.
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