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Biocatalytic (de)carboxylation of phenolic compounds: fundamentals and applications
Citation Link: https://doi.org/10.15480/882.1377
Publikationstyp
Doctoral Thesis
Date Issued
2017
Sprache
English
Author(s)
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2017-01-12
Institut
TORE-DOI
Carbon dioxide is a pivotal compound in the biotic world of metabolism and in the
abiotic world of chemical synthesis. These two worlds can merge, to some extent,
when biological catalysts for (de)carboxylations are used to accelerate chemical
conversions for industrial applications. A particular set of lyases which display this
type of activity on phenolic starting materials belong to microbial degradation
pathways. Such enzymes are hydroxybenzoic acid and phenolic acid
(de)carboxylases, which catalyze reversible carboxylations and can be differentiated
according to their regio-selectivity based on which the carboxylic group is added and
removed: ortho and para, for phenolic compounds, and beta, for hydroxycinnamic
acids. Because carbon dioxide and its hydrated form, bicarbonate, have considerably
low standard Gibbs free energies, the equilibrium of the reactions often lie strongly
on the decarboxylation side in physiological conditions. In this work, two ortho-
(de)carboxylases and one beta-(de)carboxylase were studied in the reaction directions
relevant for application. For ortho-selectivity, dihydroxybenzoic acid
(de)carboxylases –from Rhizobium sp. and Aspergillus oryzae– were studied in the
synthesis –“up-hill”– direction, which yields salicylic acids from phenols. This
reaction resembles the abiotic Kolbe-Schmitt synthesis, which is conducted at
elevated temperatures and pressures. Therefore, the establishment of an “enzymatic
counterpart” for large scale production is highly desirable in order to achieve efficient
CO2 utilization for chemical synthesis. Fundamental studies on kinetics and
thermodynamics revealed the reactions bottlenecks, allowed the proposition and
verification of a catalytic mechanism and gave new insights on the biocatalysts’
substrate spectra. The development of an amine-coupled method to use carbon
dioxide –instead of bicarbonate, the effective co-substrate usually accepted by these
biocatalysts– reveled interesting properties of ammonium ions in determining
approximately five-fold increases in reaction rates and approximately two-fold
equilibrium conversions for the carboxylation of catechol. Moreover, the analyses of
strategies to overcome the thermodynamic equilibrium are critically discussed and
demonstrate how the system may have a realistic future only on the laboratory scale.
Beta-(de)carboxylases catalyze the reversible (de)carboxylation on the C–C double
bonds of abundant and naturally occurring para-hydroxycinnamic acids yielding
para-hydroxystyrenes, which are normally obtained by using multistep synthesis,
toxic reagents and high temperatures. Therefore, these enzymes represent a
promising tool for the deoxygenation of biomass-derived feedstocks. In the present
work, a phenolic acid (de)carboxylase from Mycobacterium colombiense was studied
as biocatalyst for the decarboxylation of ferulic acid, yielding 4-vinylguaiacol, an
Food and Drug Administration-approved flavoring agent. Kinetic studies revealed
the occurrence of strong product inhibition, which was then tackled by performing
the biotransformation in two liquid-phase systems. The optimized reaction conditions
using hexane as the organic phase were demonstrated also in gram scale, affording
the target product in 75% isolated yield. A reactor concept including the integrated
product separation is also presented and discussed. In order to further exploit this
biocatalytic reaction for applications, a sequential Pd-catalyzed hydrogenation step
was carried out in the organic layer, affording 4-ethylguaiacol, another industrially
relevant flavoring agent. In gram scale, the whole reaction sequence afforded the final
product in 70% isolated yield. Both biotransformation and chemo-enzymatic
sequence were evaluated using the E-factor as a measurement of their environmental
impact; a comparison with existing synthetic paths shows how the strategies
developed in this work are promising “green” methods in view of large scale
applications.
abiotic world of chemical synthesis. These two worlds can merge, to some extent,
when biological catalysts for (de)carboxylations are used to accelerate chemical
conversions for industrial applications. A particular set of lyases which display this
type of activity on phenolic starting materials belong to microbial degradation
pathways. Such enzymes are hydroxybenzoic acid and phenolic acid
(de)carboxylases, which catalyze reversible carboxylations and can be differentiated
according to their regio-selectivity based on which the carboxylic group is added and
removed: ortho and para, for phenolic compounds, and beta, for hydroxycinnamic
acids. Because carbon dioxide and its hydrated form, bicarbonate, have considerably
low standard Gibbs free energies, the equilibrium of the reactions often lie strongly
on the decarboxylation side in physiological conditions. In this work, two ortho-
(de)carboxylases and one beta-(de)carboxylase were studied in the reaction directions
relevant for application. For ortho-selectivity, dihydroxybenzoic acid
(de)carboxylases –from Rhizobium sp. and Aspergillus oryzae– were studied in the
synthesis –“up-hill”– direction, which yields salicylic acids from phenols. This
reaction resembles the abiotic Kolbe-Schmitt synthesis, which is conducted at
elevated temperatures and pressures. Therefore, the establishment of an “enzymatic
counterpart” for large scale production is highly desirable in order to achieve efficient
CO2 utilization for chemical synthesis. Fundamental studies on kinetics and
thermodynamics revealed the reactions bottlenecks, allowed the proposition and
verification of a catalytic mechanism and gave new insights on the biocatalysts’
substrate spectra. The development of an amine-coupled method to use carbon
dioxide –instead of bicarbonate, the effective co-substrate usually accepted by these
biocatalysts– reveled interesting properties of ammonium ions in determining
approximately five-fold increases in reaction rates and approximately two-fold
equilibrium conversions for the carboxylation of catechol. Moreover, the analyses of
strategies to overcome the thermodynamic equilibrium are critically discussed and
demonstrate how the system may have a realistic future only on the laboratory scale.
Beta-(de)carboxylases catalyze the reversible (de)carboxylation on the C–C double
bonds of abundant and naturally occurring para-hydroxycinnamic acids yielding
para-hydroxystyrenes, which are normally obtained by using multistep synthesis,
toxic reagents and high temperatures. Therefore, these enzymes represent a
promising tool for the deoxygenation of biomass-derived feedstocks. In the present
work, a phenolic acid (de)carboxylase from Mycobacterium colombiense was studied
as biocatalyst for the decarboxylation of ferulic acid, yielding 4-vinylguaiacol, an
Food and Drug Administration-approved flavoring agent. Kinetic studies revealed
the occurrence of strong product inhibition, which was then tackled by performing
the biotransformation in two liquid-phase systems. The optimized reaction conditions
using hexane as the organic phase were demonstrated also in gram scale, affording
the target product in 75% isolated yield. A reactor concept including the integrated
product separation is also presented and discussed. In order to further exploit this
biocatalytic reaction for applications, a sequential Pd-catalyzed hydrogenation step
was carried out in the organic layer, affording 4-ethylguaiacol, another industrially
relevant flavoring agent. In gram scale, the whole reaction sequence afforded the final
product in 70% isolated yield. Both biotransformation and chemo-enzymatic
sequence were evaluated using the E-factor as a measurement of their environmental
impact; a comparison with existing synthetic paths shows how the strategies
developed in this work are promising “green” methods in view of large scale
applications.
Subjects
Biocatalysis
decarboxylases
kinetic modeling
salicylic acids
hydroxystyrenes
DDC Class
540: Chemie
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