Vinylation of Unprotected Phenols Using a Biocatalytic System

Article abstract of DOI:10.1002/anie.201505696

Readily available substituted phenols were coupled with pyruvate in buffer solution under atmospheric conditions to afford the corresponding para-vinylphenol derivatives while releasing only one molecule of CO₂ and water as the by-products. This transformation was achieved by designing a biocatalytic system that combines three biocatalytic steps, namely the C-C coupling of phenol and pyruvate in the presence of ammonia, which leads to the corresponding tyrosine derivative, followed by deamination and decarboxylation. The biocatalytic transformation proceeded with high regioselectivity and afforded exclusively the desired para products. This method thus represents an environmentally friendly approach for the direct vinylation of readily available 2-, 3-, or 2,3-disubstituted phenols on preparative scale (0.5 mmol) that provides vinylphenols in high yields (65-83%).

Full text of DOI:10.1002/anie.201505696

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201505696
German Edition: DOI: 10.1002/ange.201505696
Biocatalysis
Vinylation of Unprotected Phenols Using a Biocatalytic System
Eduardo Busto,* Robert C. Simon, and Wolfgang Kroutil*
Abstract: Readily available substituted phenols were coupled
with pyruvate in buffer solution under atmospheric conditions
to afford the corresponding para-vinylphenol derivatives while
releasing only one molecule of CO2 and water as the by-
products. This transformation was achieved by designing
a biocatalytic system that combines three biocatalytic steps,
Scheme 1. Biocatalytic para-selective vinylation of phenols with pyru-
vate.
À
namely the C C coupling of phenol and pyruvate in the
presence of ammonia, which leads to the corresponding
tyrosine derivative, followed by deamination and decarbox-
ylation. The biocatalytic transformation proceeded with high
regioselectivity and afforded exclusively the desired para
products. This method thus represents an environmentally
friendly approach for the direct vinylation of readily available
2-, 3-, or 2,3-disubstituted phenols on preparative scale
(0.5 mmol) that provides vinylphenols in high yields (65–
83%).
unsubstituted phenols 1 and pyruvate 2 are suitable starting
materials for a synthesis of para-vinylphenols 5 where CO2
and water are the sole by-products (Scheme 1).
In detail, the cascade process[14] was designed based on
a combination of three enzymatic reactions: In the first step,
À
the C C coupling between a phenol and pyruvate catalyzed
by a tyrosine phenol lyase (TPL)[15] leads to the formation of
l-tyrosine derivatives
3
by ammonia incorporation
T
he preparation of vinyl arenes has become a major topic in
(Scheme 2). The para selectivity is a result of the regioselec-
synthetic organic chemistry[1] as they are in great demand for
the preparation of polymers and fine chemicals.[2] Among
them, para-hydroxystyrene derivatives represent privileged
scaffolds that are widely found in advanced materials used for
the preparation of chemical and biological sensors,[3] fire
retardants, but also pharmaceuticals[4] and important organic
compounds.[5] The vinylation of unprotected phenols has been
scarcely reported, and only ortho functionalization has been
achieved thus far.[6] Methods for the preparation of para-
hydroxystyrene derivatives require a functional group in the
para position and involve the transformation of para-halo-
genated phenols with transition-metal catalysts in Heck[5b]
and Stille couplings,[7] or Knoevenagel[8] and Wittig reactions
of the corresponding aldehydes.[9] Biotechnological
approaches are limited to the decarboxylation of cinnamic
acid derivatives prepared from the corresponding alde-
hydes.[10]
Scheme 2. Three-step cascade process for the para vinylation of
phenols. FAD=ferulic acid decarboxylase, TAL =tyrosine ammonia
lyase, TPL=tyrosine phenol lyase.
tivity of the TPL. In the second concurrent step, deamination
of 3 by a tyrosine ammonia lyase (TAL)[16] provides coumaric
acid derivative 4 and ammonia. The ammonia is reused in the
first step. Finally, decarboxylation of 4 catalyzed by a ferulic
acid decarboxylase (FAD)[17] leads to the desired vinylated
phenol 5.
To the best of our knowledge, no single enzyme is able to
catalyze the para vinylation of phenols; therefore, a biocata-
lytic system,[11,12] that is, an in vitro enzyme cascade combin-
ing enzymes from different organisms, was designed to
achieve the vinylation of phenols in buffer solution. A
biocatalytic retrosynthetic analysis[13] revealed that para-
When testing possible enzymes we found that the
recombinant tyrosine phenol lyase from Citrobacter freundii
and its variant M379V[15c] were most suitable for the C C
À
[*] Dr. E. Busto,[+] Dr. R. C. Simon, Prof. Dr. W. Kroutil
Department of Chemistry, University of Graz, NAWI Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: bbusto@ucm.es
coupling step; we chose to use the variant M379V for further
experiments because of its broader substrate scope.[15c] This
biocatalyst was employed as a cell-free extract. The most
promising results for the deamination step were achieved with
a TAL from Rhodobacter spharoides overexpressed in E. coli,
which was used as a freeze-dried cell preparation.[16b] The
ferulic acid decarboxylase from Enterobacter sp. was overex-
pressed in E. coli and also used as a freeze-dried cell
preparation for the final decarboxylation step.[17a] We
wolfgang.kroutil@uni-graz.at
[+] Present address: Department of Organic Chemistry I
Universidad Complutense de Madrid
28040 Madrid (Spain)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201505696.
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Table 1: Vinylation of 2-fluorophenol (1a) at various ammonium con-
assumed that under our reaction conditions, this final step
would shift the overall reaction sequence to completion.
To identify a suitable pH value for the three-step cascade
process, the pH ranges of the three individual catalysts were
compared (see the Supporting Information): The TPL as well
as the TAL achieved the highest conversions at more alkaline
pH values (pH 8–10 for TPL and pH 10 for TAL) whereas the
decarboxylase preferred a pH range of 6–8. As the optimal
pH values of the three enzymes are not the same, a compro-
mise had to be found; For this purpose, we studied the
dependence of the overall cascade process on the pH value
using 2-fluorophenol (1a) as the substrate (Figure 1). The
centrations.[a]
Entry
NH4+ [mm]
2 [mm]
5a[b] [%]
1
2
3
4
5
12
23
46
180
180
46
46
46
46
23
55
72
93
>99
88
[a] Reaction conditions: 1a (23 mm), pyruvate, NH4Cl, PLP (0.04 mm),
TPL M379V (1.2 U, 4 mg cell-free extract), TAL (0.18 U, 20 mg freeze-
dried E. coli/TAL), FAD (10.5 U, 5 mg freeze-dried E. coli/FAD), potas-
sium phosphate buffer (50 mm, pH 8), Et2O (5% v/v), 308C, 16 h,
850 rpm. [b] Determined by reverse-phase HPLC analysis.
Figure 1. Conversion into vinylphenol 5a at various pH values. Reac-
tion conditions: 1a (23 mm), pyruvate (46 mm), NH4Cl (180 mm),
buffer (50 mm; potassium phosphate for pH 7–8, CHES for pH 9–10),
PLP (0.04 mm), TPL (M379V, 1.2 U, 4 mg cell-free extract), TAL
(0.18 U, 20 mg freeze-dried E. coli/TAL), FAD (10.5 U, 5 mg freeze-
dried E. coli/FAD), Et2O (5% v/v), 308C, 850 rpm.
Figure 2. Time course for the biocatalytic vinylation of 2-fluorophenol
(1a) into 2-fluoro-4-vinylphenol (5a). Reaction conditions: 1a
(23 mm), pyruvate (46 mm), NH4Cl (180 mm), PLP (0.04 mm), TPL
M379V (1.2 U, 4 mg cell-free extract), TAL (0.18 U, 20 mg freeze-dried
E. coli/TAL), FAD (10.5 U, 5 mg freeze-dried E. coli/FAD), potassium
phosphate buffer (50 mm, pH 8), Et2O (5% v/v), 308C, 850 rpm.
highest concentrations of 5a were obtained at pH 8 in
phosphate buffer and at pH 9 in CHES buffer. Even though
the two results are comparable, further experiments were
performed at pH 8 in the phosphate buffer as this buffer is less
expensive and thus more suitable for future applications. The
slower product formation that was observed at pH 7 was most
likely due to the lower activity of the two lyases (TPL, TAL)
under these conditions (see the Supporting Information for
a detailed reaction course). On the other hand, the slower
formation of 5a at pH 10 and the accumulation of coumaric
acid 4a are due to the low activity of the FAD at high pH (see
the Supporting Information).
Reactions were performed in a solution containing Et2O
(5% v/v) as co-solvent[15c] as slightly better results were
obtained compared with the reactions in the absence of this
additive or with a water-miscible co-solvent such as DMSO
(see the Supporting Information).
In the next optimization step, the equivalents of pyruvate
and ammonium chloride were varied. When 0.5 equivalents
of NH4Cl were used, the cascade process led to the formation
of vinylphenol 5a in 55% within 16 hours (Table 1, entry 1).
When the amount of NH4Cl was increased to 180 mm
(entries 2–4) at a pyruvate concentration of 46 mm, the
desired product 5a was formed in > 99% conversion when
measured after 16 hours. The formation of undesired side
products, for example, by ortho vinylation, was not detected.
Furthermore, polymerization of the vinylphenols is not an
issue under these reaction conditions.
Following the bio-vinylation of 1a to 5a over time at pH 8
and 308C revealed that the concentration of tyrosine deriv-
ative 3a reached a maximum after 60 min (Figure 2). The
permanently low concentration of coumaric acid derivative
4a indicated that the decarboxylation step is significantly
faster than the previous step under the conditions employed.
The concentration of the final vinylphenol 5a increased
linearly for the first three hours; then, its formation slowed
down, and the reaction reached completion after eight hours.
The reaction course indicates that the deamination reaction
catalyzed by the TAL is the rate-determining step of the
cascade process under these conditions as tyrosine derivative
3a was accumulated in the reaction mixture. This is in
agreement with the activity values measured for the three
catalysts (see the Supporting Information).
The scope of the method was explored with a variety of 2-
or 3-substituted phenols (Table 2). Aside from 2-fluorophenol
(1a), phenols with a chloro (1b), bromo (1c), or methyl (1d)
substituent in the 2-position were also successfully vinylated
with > 99% conversion (entries 1–5). The biovinylation was
also successful for 3-substituted phenols such as 3-fluoro- (1e)
or 3-chlorophenol (1 f). Furthermore, 2,3-disubsituted phe-
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Table 2: Preparative-scale experiments for the biocatalytic vinylation of
as 2,3-disubstituted phenols (1g and 1h) corroborated the
potential of our method, as the corresponding vinylated
phenols were isolated in 65–83% yield (Table 2).
phenols.[a]
In conclusion, para-unsubstituted phenol derivatives were
transformed into the corresponding para-vinylphenols in
À
a three-step biocatalytic cascade process comprising a C C
coupling, deamination, and decarboxylation. The overall
reaction required only pyruvate as a stoichiometric reagent,
leading to the formation of CO2 and water as the sole by-
products. The transformations were performed at substrate
concentrations of 10–46 mm leading to > 99% conversion
Entry
1
Substrate
1 [mm]
Product 5
Conv.[b] [%]
1a
23
>99 (69)
within 8–24 h and good yields of isolated products (65–83%).
[19]
À
A novel method for C C bond formation and an environ-
2[d]
1a
1b
1c
1d
46
23
10
10
>99 (71)
>99 (68)
>99[c]
mentally friendly and mild route for the efficient preparation
of para-vinylphenol derivatives, intermediates that are
required for the synthesis of various advanced materials,
have thus been developed.
3
4
Experimental Section
5
>99[c]
Representative procedure for the para vinylation of phenol 1a:
Recombinant TPL M379V from C. freundii (15 U, 50 mg cell-free
extract), TAL from R. spharoides (3.68 U, 400 mg freeze-dried E. coli/
TAL cells), and FAD from Enterobacter sp. (105 U, 50 mg freeze-
dried E. coli/FAD cells) were rehydrated in a potassium phosphate
buffer (50 mm, 180 mm NH4Cl, 0.04 mm PLP, pH 8, 10 mL) for 10 min
at 308C and 120 rpm. Then, phenol 1a (42 mL, 0.46 mmol, 46 mm)
dissolved in Et2O (500 mL) and pyruvate (101.2 mg, 0.92 mmol,
92 mm) were added to the mixture, and the reaction was incubated
for 24 h at 218C and 170 rpm. The reaction was stopped by the
addition of an aqueous saturated NH4Cl solution (5 mL) and
extracted with EtOAc (3 ” 15 mL). The combined organic phases
were dried (Na2SO4), and the solvent was evaporated under reduced
pressure (100 mmHg). The crude product was purified by flash
chromatography on silica gel (20% EtOAc/hexane) to afford the
corresponding vinylphenol 5a as a colorless oil (45 mg, 71%).
6[d]
1e
1 f
46
23
46
23
>99 (77)
>99 (83)
>99 (65)
>99 (65)
7
8
1g
1h
9[d,e]
[a] Reaction conditions: Phenol 1, pyruvate (2 equiv), NH4Cl (180 mm),
TPL (50 mg, 15 U, cell-free extract), TAL (200 mg, 1.84 U, E. coli whole
cells), FAD (50 mg, 105 U, E. coli whole cells), potassium phosphate
buffer (pH 8, 10 mL, 50 mm), Et2O (5% v/v), 308C, 120 rpm, 24 h.
[b] Neither starting material 1 nor the intermediates l-3 or 4 were
detected. Conversions were determined by reverse-phase HPLC analysis.
Yields after flash column chromatography are given in parentheses.
[c] Experiment performed on analytical scale, see the Supporting
Information. [d] Double amounts of the three biocatalysts were used.
[e] Reaction time: 48 h.
Acknowledgements
E.B. received funding from the European Commission
through a Marie Curie Actions-Intra-European Fellowship
(IEF) as part of the “BIOCASCADE” project (FP7-
PEOPLE-2011-IEF, PIEF-GA-2011-298030). We also thank
COST Action CM1303, “Systems Biocatalysis”.
À
Keywords: biotransformations · cascade reactions · C
C bond formation · enzyme catalysis · vinylation
nols, such as 2,3-difluoro- (1g) and 2-fluoro-3-chlorophenol
(1h), were efficiently transformed into the corresponding
para-vinylphenols (entries 8 and 9). Several of these vinylated
phenols have not been prepared before (5a and 5c) or have
not even been described (5 f and 5h),[18] which clearly shows
that the simple method presented in this manuscript expands
the range of vinylphenols that can be prepared.
The para vinylation could also be carried out on prepa-
rative scale, leading, for instance, to the isolation of fluori-
nated product 5a in 71% yield after filtration through silica
gel when the reaction was performed at a phenol concen-
tration of 46 mm (entry 2). Preparative transformations of 2-
chlorophenol (1b), 3-substituted phenols (1e and 1 f), as well
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Received: June 20, 2015
Published online: July 29, 2015
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