Regioselective enzymatic carboxylation of phenols and hydroxystyrene derivatives

Article abstract of DOI:10.1021/ol300385k

The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the β-carbon atom of styrenes forming (E)-cinnamic acids.

Full text of DOI:10.1021/ol300385k

ORGANIC
LETTERS
2012
Vol. 14, No. 8
1974–1977
Regioselective Enzymatic Carboxylation
of Phenols and Hydroxystyrene
Derivatives
Christiane Wuensch,† Silvia M. Glueck,† Johannes Gross,† Dominik Koszelewski,†
Markus Schober,‡ and Kurt Faber*,‡
Austrian Centre of Industrial Biotechnology, c/o Department of Chemistry, Organic &
Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Kurt.Faber@Uni-Graz.at
Received February 16, 2012
ABSTRACT
The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective
fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at
the β-carbon atom of styrenes forming (E)-cinnamic acids.
In order to alleviate the predominant dependence of the
chemical industry from petroleum-based platform
intermediates,1 the development of CO2-fixation reactions
would allow conversion of a problematic waste gas into a
useful carbon source for the production of chemicals.2 The
biggest obstacle to surmount is the low energy level of
CO2. Carboxylation is facilitated by using high-energy
starting materials, such as H2, organometallics,3 unsatu-
rated compounds(alkenes, alkynes, allenes4), or epoxides.5
Alternatively, carboxylation toward low-energy products
containing carbon at a high oxidation state yields carbon-
ates, carbamates, carboxylic acids, and esters or lactones.
Although the number of processes based on chemical CO2-
fixation is small, the volumes of production (e.g., urea,
KolbeꢀSchmitt reaction for phenol-carboxylation6) are
impressive.7 Despite these isolated success stories, the use
of CO2 as raw material for organic synthesis is still heavily
underdeveloped.2,8 In this context, the biocatalytic CO2-
fixation catalyzed by (de)carboxylases holds great poten-
tial. Analysis of biological CO2-fixation reveals that the
major high-energy pathways9 are more substrate-specific
and thus are less likely to be adapted to non-natural
organic compounds.10 In contrast, detoxification path-
ways are more promising, because the removal of a multi-
tude of toxinsbya single broad-spectrum enzymeenhances
the survival of the living system. Since carboxylation is a
† Austrian Centre of Industrial Biotechnology.
‡ Department of Chemistry, Organic & Bioorganic Chemistry.
(1) The major intermediates derived from crude oil encompass
methanol, alkenes (ethene, propene, butene, butadiene), and aromatics
(benzene, toluene, xylenes).
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recycled” by these processes to date.
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r
10.1021/ol300385k
Published on Web 04/03/2012
2012 American Chemical Society

redox-independent process, it is ideally suited for the
detoxification of phenolics in anaerobic organisms,10,11
which represents a biocatalytic equivalent to the Kolbeꢀ
Schmitt reaction,6 which requires pressurized CO2 and
elevatedtemperatures(120ꢀ300°C)and oftensuffersfrom
incomplete regioselectivities.
Although the biodegradation of phenolic compounds
via carboxylation by whole microbial cells is reasonably
well understood,11b the respective enzymes were predomi-
nantly investigated for their (downhill) decarboxylation
activities.12 In contrast, only limited data are available on
the enzymatic carboxylation of (hetero)aromatics using
(de)carboxylases running in the reverse (synthetic)
direction:
was also able to convert phenol, 1,2-dihydroxyben-
zene, and m-aminophenol at very low rates.20
(iv) In contrast to the carboxylases mentioned above,
which strictly depend on the presence of a phenolic
functional group in the substrate, electron-excess
heteroaromatic species (e.g., pyrrole, indole) were
carboxylated at position 2 or 3, respectively, by
pyrrole-2- carboxylate decarboxylase (max. con-
version 80%21) and indole-3-carboxylase (max.
conversion 34%22) (Scheme 1). Unfortunately, both
enzymes appear to be highly substrate specific and
only tolerate minimal structural variations.
(i) p-Carboxylation of phenol yielding p-hydroxyben-
zoic acid is catalyzed by phenylphosphate
carboxylase,11b,13 which requires activation of the
substrate by (energy-consuming) phosphorylation
with ATP prior to carboxylation. In contrast, 4-
hydroxybenzoate decarboxylase was found to cata-
lyze the direct (reverse) carboxylation of phenol at a
slow rate14 (max. conversion 19%15).
(ii) The regio-complementary o-carboxylation of phenol
was catalyzed by salicylic acid decarboxylase with a
respectable conversion of 27%.16 Most interestingly,
the carboxylation of m-aminophenol selectively gave
the antituberculostatic agent p-aminosalicylic acid
with 70% conversion.17
(iii) 1,2-Dihydroxybenzene (catechol) was selectively
carboxylated at the o-position by 3,4-dihydroxy-
benzoate decarboxylase in up to 28% conversion.18
The 1,3-analog (resorcinol) was carboxylated at the
2-position by 2,6-dihydroxybenzoate decarboxylase
in up to 48% conversion.19 Although the enzyme was
completely regioselective on its ’natural’ substrate, it
Scheme 1. Regio-complementary Enzymatic Carboxylation of
Phenols and Hydroxystyrene Derivatives
In addition to the benzoic acid (de)carboxylases dis-
cussed above, which catalyze the carboxylation of an
aromatic system, phenolic acid decarboxylases23 act on
the side chain of hydroxycinnamic acids yielding styrenes.
The reverse carboxylation activity of the latter enzymes is
unknown. Based on the limited structural data available to
date, both types of enzymes act through completely dif-
ferent mechanisms: Whereas benzoic acid decarboxylases
are metal-dependent and requireacatalyticallyactiveZn2þ
in the active site,24 the mechanism of phenolic acid de-
carboxylases proceeds via general (metal-independent)
acidꢀbase catalysis.25
In order to ensure the practical applicability of the
enzymatic carboxylation, we avoided oxygen-sensitive
enzymes13a,14a,14b,15 and thus selected (de)carboxylases,
which are known to be oxygen-stable.
(11) (a) Brackmann, R.; Fuchs, G. Eur. J. Biochem. 1993, 213, 563–
571. (b) Boll, M.; Fuchs, G. Biol. Chem. 2005, 386, 989–997. (c) Gobson,
J.; Harwood, C. S. Annu. Rev. Microbiol. 2002, 56, 345–369. (d)
Ackermann, L. Angew. Chem., Int. Ed. 2011, 50, 3842–3844.
(12) Liu, A.; Zhang, H. Biochemistry 2006, 45, 10407–10411.
(21) (a) Wieser, M.; Yoshida, T.; Nagasawa, T. J. Mol. Catal. B:
Enzym. 2001, 11, 179–184. (b) Wieser, M.; Fujii, N.; Yoshida, T.;
Nagasawa, T. Eur. J. Biochem. 1998, 257, 495–499. (c) Wieser, M.;
Yoshida, T.; Nagasawa, T. Tetrahedron Lett. 1998, 39, 4309–4310.
(22) Yoshida, T.; Fujita, K.; Nagasawa, T. Biosci. Biotechnol. Bio-
chem. 2002, 66, 2388–2394.
(23) These enzymes are also denoted as hydroxycinnamic acid de-
carboxylase, ferulic acid decarboxylase, or p-coumaric acid decarbox-
ylase. (a) Rodriguez, H.; Landete, J. M.; Curiel, J. A.; de las Rivas, B.;
Mancheno, J. M.; Munoz, R. J. Agric. Food Chem. 2008, 56, 3068–3072.
(b) Zago, A.; Degrassi, G.; Bruschi, C. V. Appl. Environ. Microbiol. 1995,
61, 4484–4486. (c) Cavin, J.-F.; Barthelmebs, L.; Divies, C. Appl.
Environ. Microbiol. 1997, 63, 1939–1944. (d) Gu, W.; Li, X.; Huang,
J.; Duan, Y.; Meng, Z.; Zhang, K.-Q.; Yang, J. Appl. Microbiol.
Biotechnol. 2011, 89, 1797–1805. (e) Prim, N.; Pastor, F. I. J.; Diaz, P.
Appl. Microbiol. Biotechnol. 2003, 63, 51–56.
(24) Goto, M.; Hayashi, H.; Miyahara, I.; Hirotsu, K.; Yoshida, M.;
Oikawa, T. Crystal structures of nonoxidative Zn-dependent 2,6-dihy-
droxybenzoate (γ-resorcylate) decarboxylase from Rhizobium sp. strain
Mtp-10005, pdb accession number entry 2DVT_A.
(25) (a) Matte, A.; Grosse, S.; Bergeron, H.; Abokitse, K.; Lau,
P. C. K. Acta Crystallogr., Sect. F 2010, F66, 1407–1414. (b) Gu, W.;
Yang, J.; Lou, Z.; Liang, L.; Sun, Y.; Huang, J.; Li, X.; Cao, Y.; Meng,
Z.; Zhang, K.-Q. PLoS ONE 2011, 6, e16262, DOI: 10.1371/journal.
pone.0016262. (c) Rodriguez, H.; Angulo, I.; de las Rivas, B.; Campillo,
N.; Paez, J. A.; Munoz, R.; Mancheno, J. M. Proteins 2010, 78, 1662–
1676.
(13) (a) Aresta, M.; Quaranta, E.; Liberio, R.; Dileo, C.; Tommasi, I.
Tetrahedron 1998, 54, 8841–8846. (b) Dibenedetto, A.; Lo Noce, R.;
Pastore, C.; Aresta, M.; Fragale, C. Environ. Chem. Lett. 2006, 3, 145–
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148. (c) Schuhle, K.; Fuchs, G. J. Bacteriol. 2004, 186, 4556–4567.
(14) (a) Liu, J.; Zhang, X.; Zhou, S.; Tao, P.; Liu, J. Curr. Microbiol.
2007, 54, 102–107. (b) He, Z.; Wiegel, J. Eur. J. Biochem. 1995, 229, 77–
82. (c) Lupa, B.; Lyon, D.; Shaw, L. N.; Sieprawska-Lupa, M.; Wiegel, J.
Can. J. Microbiol. 2008, 54, 75–81.
(15) Matsui, T.; Yoshida, T.; Hayashi, T.; Nagasawa, T. Arch.
Microbiol. 2006, 186, 21–29.
(16) Kirimura, K.; Gunji, H.; Wakayama, R.; Hattori, T.; Ishii, Y.
Biochem. Biophys. Res. Commun. 2010, 394, 279–284.
(17) Kirimura, K.; Yanaso, S.; Kosaka, S.; Koyama, K.; Hattori, T.;
Ishii, Y. Chem. Lett. 2011, 40, 206–208.
(18) (a) He, Z.; Wiegel, J. J. Bacteriol. 1996, 178, 3539–3543. (b)
Yoshida, T.; Inami, Y.; Matsui, T.; Nagasawa, T. Biotechnol. Lett. 2010,
32, 701–705.
(19) This enzyme is also termed γ-resorcylate decarboxylase. (a)
Yoshida, M.; Fukuhara, N.; Oikawa, T. J. Bacteriol. 2004, 186, 6855–
6863. (b) Ishii, Y.; Narimatsu, Y.; Iwasaki, Y.; Arai, N.; Kino, K.;
Kirimura, K. Biochem. Biophys. Res. Commun. 2004, 324, 611–620. (c)
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2004, 181, 391–397.
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Org. Lett., Vol. 14, No. 8, 2012
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Table 1. Enzymatic Carboxylation of Phenols and Styrene Derivatives Using Benzoic and Phenolic Acid Decarboxylasesa
a Reaction conditions: whole lyophilized E. coli cells containing overexpressed (de)carboxylase (30 mg), substrate (10 mM), KHCO3 (3 M), Pi buffer
(pH 8.5, 100 mM), 30°C, 120 rpm, 24 h. b 2,3-DHBD_Ao = 2,3-dihydroxybenzoate decarboxylase (Aspergillus oryzae).26 c 2,6-DHBD_Rs = 2,6-
dihydroxybenzoate decarboxylase (Rhizobium sp.).27 d SAD_Tm = salicylic acid decarboxylase (Trichosporon moniliiforme).28 e PAD_Lp = phenolic
acid decarboxylase (Lactobacillus plantarum).29 f PAD_Ba = phenolic acid decarboxylase (Bacillus amyloliquefaciens).30
The following candidates were cloned and (over)
expressed in E. coli BL21 (DE3) as host: 2,3-Dihydroxy-
benzoate decarboxylase from Aspergillus oryzae (2,3-
DHBD_Ao),26 2,6-dihydroxybenzoate decarboxylase from
Rhizobium sp. (2,6-DHBD_Rs),27 salicylic acid decarbox-
ylase from Trichosporon moniliiforme (SAD_Tm),28 pheno-
lic acid decarboxylases from Lactobacillus plantarum and
Bacillus amyloliquefaciens (PAD_Lp,29 PAD_Ba).30 Car-
boxylation reactions were performed in glass vials using
whole lyophilized E. coli cells (showing activities of 1.3 U/mg
(PDC_Lp)) in phosphate buffer. After addition of sub-
strate (10 mM) and KHCO3 (3 M, pH 8.5), the samples
were shakenat30°C and 120 rpm. After extractiveworkup
(24 h) reversed-phase HPLC was used to determine the
conversion. Blank-experiments using empty E. coli host
cells ensured the absence of competing (de)carboxylation
activities (see Supporting Information).
For the mapping of the substrate tolerance, the candi-
date enzymes were tested using a representative set of
phenols (1aꢀ3a), as well as dihydroxybenzene (4aꢀ6a)
and styrene derivatives (7aꢀ9a) bearing electron-donating
or -withdrawing groups. Since the application of CO2-
pressurized reaction conditions (up to 2 bar) did not have
any significant effects on the conversion, it was assumed
(26) NCBI Reference GI: 94730373. Santha, R.; Appaji Rao, N.;
Vaidyanathan, C. S. Biochim. Biophys. Acta 1996, 1293, 191–200.
(27) NCBI Reference GI: 116667102; see ref 19a.
(28) NCBI Reference GI: 225887918; see ref 16.
(29) NCBI Reference GI: 300769086; see ref 25c.
(30) NCBI Reference GI: 308175189.
(31) Lindskog, S. Pharmacol. Ther. 1997, 74, 1–20.
1976
Org. Lett., Vol. 14, No. 8, 2012

that the cosubstrate more likely is (bi)carbonate rather
than CO2, which equilibrates rather slowly. Attempts
to speed up CO2/(bi)carbonate equilibration using car-
bonic anhydrase failed, which was most likely caused
by inhibition of carbonic anhydrase by the phenolic
substrates.31 Variation of the (hydrogen) carbonate
concentration within a range of 0.1ꢀ4.0 M revealed
that a 3 M KHCO3 solution buffered at pH 8.5 gave an
optimum in terms of reaction rate and conversion. All
products were identified by comparison with authentic
reference materials via coinjection on HPLC/UV and/
or NMR.
The results depicted in Table 1 reveal the following
trends: overall, all benzoic acid decarboxylases showed
an exclusive regioselectivity for the carboxylation of the
aromatic system in the o-position of the ’directing’ pheno-
lic group;32 no trace of regioisomeric p-carboxylation
products, which are often formed in the KolbeꢀSchmitt
reaction, could be detected. In the absence of a free
o-position (9a), no reaction occurred. Surprisingly, no clear
preference for the corresponding ’natural’ substrates (1a
with SAD_Tm, 4a with 2,3-DHBD_Ao, 5a with 2,6-
DHBD_Rs) could be detected, and several cross-activities
within the same range of conversion (1a with 2,3-
DHBD_Ao, 4a with 2,6-DHBD_Rs and SAD_Tm, 5a
with 2,3-DHBD_Ao and SAD_Tm) suggest that the sub-
strate tolerance of these enzymes was remarkably broad.
Only 2,6-DHBD_Rs was inactive on phenol (1a). Fortu-
nately, also non-natural substrates, such as 1-naphthol
(2a) and m-aminophenol (3a), were regioselectively car-
boxylated to the corresponding o-products 2b and 3b in up to
62 and 80% conversion, respectively. Surprisingly, even olive-
tol (6a) bearing a sterically demanding C5-alkyl chain was
regioselectively carboxylated to yield 6b in up to 56% conver-
sion. In contrast, steric effects seem to play a major role for the
o-carboxylation of styrene-type substrates: whereas 7a was
carboxylated in up to 58% conversion, 8a gave 29% conver-
sion at best with 2,3-DHBD_Ao and SAD_Tm; 2,6-
DHBD_Rs was unreactive. In the absence of a free o-position
(9a), no reaction took place, and the vinylic group always
remained untouched. Overall, whereas 2,6-DHBD_Rs
showed a clear preference for dihydroxybenzene derivatives,
2,3-DHBD_Ao also accepted simple phenols.
In contrast to benzoic acid decarboxylases, both phe-
nolic acid decarboxylases PAD_Lp and PAD_Ba showed
complete regio-complementary behavior: The aromatic
system remained completely unaffected, and carboxyla-
tion selectively occurred at the β-carbon atom of the side
chain by forming the corresponding (E)-cinnamic acid
derivatives (7cꢀ9c) in low to modest conversion (cmax.
30%). This biocatalytic transformation is remarkable,
because (to the best of our knowledge) it does not have
any direct counterpart in the arsenal of chemical catalysis,
the closest analogue being the Ni-catalyzed hydrocarbox-
ylation of styrenes,33 which forms 2-arylpropionic acids,
rather than cinnamic acid derivatives. Detailed studies on
the exploitation of the regio-complementary carboxylation
of styrene derivatives are underway.
Acknowledgment. This study was performed in coop-
eration within the strategic research programme of the
Austrian Centre of Industrial Biotechnology (ACIB,
funded through the FFG-COMET-Program) and the
DK Molecular Enzymology (Project W9), and support
by the FWF, BMWFJ, BMVIT, SFG, Standortagentur
Tirol and ZIT is gratefully acknowledged. Klaus Zangger
(University of Graz, Department of Chemistry) is thanked
for his great support in NMR spectroscopy. Byung-Gee
Kim (School of Chemical and Biological Engineering,
Seoul National University, Seoul, South Korea) is cor-
dially thanked for the generous donation of phenolic acid
decarboxylase plasmids.
Supporting Information Available. Gene sequences,
cloning and overexpression of (de)carboxylases, SDS-
PAGE analysis, general carboxylation procedure, blank
experiments using empty host cells, synthesis of sub-
strates and reference materials, analytical procedures,
optimization of carbonate concentration, CO2 pressure
experiments, and NMR spectra are given in the electronic
Supporting Information. This material is available free of
charge via the Internet at http://pubs.acs.org.
(32) Preliminary data indicate that the phenolic moiety seems to play
an important role in catalysis, since the nonphenolic substrates tested
were unreactive (data not shown).
(33) (a) Williams, C. M.; Johnson, J. B.; Rovis, T. J. Am. Chem. Soc.
2008, 130, 14936–14937. (b) Zhang, Y.; Riduan, S. N. Angew. Chem., Int.
Ed. 2011, 50, 6210–6212.
The authors declare no competing financial interest.
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