Adsorbent-Based Downstream-Processing of the Decarboxylase-Based Synthesis of 2,6-Dihydroxy-4-methylbenzoic Acid

Article abstract of DOI:10.1021/acs.oprd.8b00104

In this case study the regioselective enzymatic carboxylation of 3,5-dihydroxytoluene (orcinol) using the nonoxidative 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD-Ao), followed by an adsorbent-based downstream approach, has been investigated. The product 2,6-dihydroxy-4-methylbenzoic acid (DHMBA) was herein purified by an adsorption-desorption cycle and subsequently obtained with purities >99% without a full elimination of the excess bicarbonate from its reaction solution. Ten adsorbent resins were studied in respect of their ability to recover the product from the reaction solution, whereas the strong anion exchange resin Dowex 1x2 in its chloride form showed affinities >99%, even at bicarbonate concentrations of >3 mol·L^-1. Desorption from loaded resin was carried out by a 2 mol·L^-1 HCl/acetone mixture, followed by product crystallization during acetone evaporation. This presented concept does not require a final column preparation step and improves the overall atom efficiency of the biocatalytic reaction system.

Full text of DOI:10.1021/acs.oprd.8b00104

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Full Paper
Adsorbent-based downstream-processing of the decarboxylase-
based synthesis of 2,6-dihydroxy-4-methylbenzoic acid
Lars-Erik Meyer, Katharina Plasch, Udo Kragl, and Jan von Langermann
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00104 • Publication Date (Web): 29 Jun 2018
Downloaded from http://pubs.acs.org on June 30, 2018
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Adsorbentꢀbased downstreamꢀprocessing of the
decarboxylaseꢀbased synthesis of
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2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid
Lars-Erik Meyer1, Katharina Plasch2, Udo Kragl1,3,# and Jan von Langermann1,*
1 University of Rostock, Institute of Chemistry, AlbertꢀEinsteinꢀStr. 3a, 18051 Rostock,
Germany
2 University of Graz, Organic & Bioorganic Chemistry, Heinrichstrasse 28, Aꢀ8010 Graz,
Austria
3
Faculty for Interdisciplinary Research, Department Life, Light and Matter, University of
Rostock, 18051 Rostock, Germany
KEYWORDS
Downstreamꢀprocessing (DSP); orthoꢀbenzoic acid decarboxylases; biocatalysis;
carboxylation; ionꢀexchange resin
ABSTRACT
In this case study the regioselective enzymatic carboxylation of 3,5ꢀdihydroxytoluene
(orcinol) using the nonoxidative 2,3ꢀdihydroxybenzoic acid decarboxylase from Aspergillus
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oryzae (2,3ꢀDHBD_Ao), followed by an adsorbentꢀbased downstream approach, has been
investigated. The product 2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid (DHMBA) was herein purified
by an adsorptionꢀdesorptionꢀcycle and subsequently obtained with purities >99% without a
full elimination of the excess bicarbonate from its reaction solution. Ten adsorbent resins
were studied in respect of their ability to recover the product from the reaction solution,
whereas the strong anion exchange resin Dowex 1x2 in its chlorideꢀform showed affinities
>99%, even at bicarbonate concentrations of >3 mol·Lꢀ1. Desorption from loaded resin was
carried out by a 2 mol·Lꢀ1 HCl/acetone mixture, followed by product crystallization during
acetone evaporation. This presented concept does not require a final column preparation step
and improves overall atom efficiency of the biocatalytic reaction system.
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1. INTRODUCTION
The synthesis of hydroxybenzoic acids was originally introduced into industrial scale by
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th
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Hermann Kolbe and Rudolf Schmitt at the end of the 19 century. The KolbeꢀSchmitt
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process enables access to important aromatic carboxylic acids such as pharmaceuticals,
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agrochemicals and building blocks for organic synthesis. Unfortunately, this procedure
frequently suffers from unsatisfactory regioselectivity and requires harsh reaction conditions
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such as high temperatures and CO2 pressures (120ꢀ300 °C, 20ꢀ100 atm). Further
disadvantages may include the preparation and isolation of completely dry phenoxide from
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the corresponding phenol, depending on the phenolꢀsubstrate. Despite these challenges, the
KolbeꢀSchmitt reaction is still widely used in industry, especially for the syntheses of salicylic
acid, which is a precursor of acetylsalicylic acid and more well known as Aspirin® by Bayer
AG.
However, in recent years multiple investigations were carried out to circumvent the
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disadvantages of the KolbeꢀSchmittꢀreaction, e.g. by using (transition) metal catalysis,
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microwaveꢀassisted reactions and ionic liquids. A further promising biocatalytic alternative
is the use of nonoxidative (de)carboxylases, which catalyze the reversible
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introduction/removal of a carboxylic group from organic compounds. This can be
synthetically applied for the regioselective orthoꢀ and paraꢀcarboxylation of small or
mediumꢀsized phenol derivatives using bicarbonate as CO2ꢀsource.10 Recently also more
complex (poly)phenolic substrates were carboxylated using orthoꢀ(de)carboxylases
2,3ꢀdihydroxybenzoic acid (de)carboxylase from Aspergillus oryzae (2,3ꢀDHBD_Ao),
2,6ꢀdihydroxybenzoic acid (de)carboxylase from Rhizobium sp. (2,6ꢀDHBD_Rs) and salicylic
acid decarboxylase from Trichosporon moniliiforme (SAD_Tm) (Scheme 1).14
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Scheme 1. Regioselective enzymatic carboxylation of phenols using bicarbonate as a CO2ꢀ
source leading to a wide range of products.
In strong contrast to the original KolbeꢀSchmitt reaction, significantly less harsh reaction
conditions are required for such a biocatalytic carboxylation reaction with a reaction
temperature of only 30 °C at or near atmospheric pressure (using bicarbonate as CO2ꢀsource).
Unfortunately, the reaction equilibrium of the biocatalytic alternative is similarly positioned
towards the phenolꢀcompounds.11 Hence, strategies to push the equilibrium of
(de)carboxylaseꢀcatalyzed reactions toward the formation of carboxylation products are
required.12 Here, bicarbonate or CO2 is usually applied in excess and recent studies also
suggest the use of amines/quaternary ammonium salts for a (partial) equilibrium displacement
towards the carboxylic acid.13 In addition, downstreamꢀprocessing of (de)carboxylase
reactions is still a challenge and, to the best of our knowledge, a total elimination of the
excess of bicarbonate (see above) via acid addition is still the preferred route. A column
chromatography is still subsequently required to remove remaining substrate and further
impurities from the biocatalyst preparation, e.g. from the cell preparation method in LB
medium.14
In this work, we report the development of an alternative adsorbentꢀbased downstream
approach for a (de)carboxylaseꢀcatalyzed reaction to facilitate selective product isolation from
the reaction mixture without removing the excess of bicarbonate (Fig. 1). This approach was
investigated for the highly stereoselective 2,3ꢀDHBD_Aoꢀcatalyzed (2,3ꢀdihydroxybenzoate
decarboxylase from Aspergillus oryzae,15 NCBI Reference GI: 94730373) orthoꢀ
carboxylation of 3,5ꢀdihydroxytoluene (orcinol) to 2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid
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(DHMBA) (Scheme 2).16 DHMBA is a valuable precursor for the synthesis of intermediates
for novel inhibitors to target acetohydroxy acid synthase (AHAS; a major target for herbicide
discovery),17 the synthesis of Debromohamigeran E,18 and further antiꢀrheumatic agents and
immunosuppressants (Scheme 2).
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Fig. 1. Schematic representation of adsorbentꢀbased scaleꢀup of the decarboxylaseꢀbased
synthesis of 2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid (DHMBA) leading to pure product without
further purification steps.
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Scheme 2. Enzymatic orthoꢀcarboxylation of orcinol catalyzed by 2,3ꢀDHBD_Ao using
bicarbonate as CO2ꢀsource (A) and subsequent products [(B) Qu et al.17, (C) Blaisdell et al.18
(D) Hoffmann et al.19].
,
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2. MATERIALS AND METHODS
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General
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All compounds were received from commercial suppliers in highest available purity
(SigmaꢀAldrich, Alfa Aesar, TCI, Fluka) and used as received. Ultrapure water was produced
with an ‘Ultra Clear Reinstwassersystem’ by SG Water (now Evoqua, Guenzburg, Germany)
and used throughout this study. 2,3ꢀDihydroxybenzoic acid decarboxylase from Aspergillus
oryzae (2,3ꢀDHBD_Ao) was cloned and overexpressed in E. coli BL21 (DE3) as previously
described.20
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Resin beads
Resins Diaion PA312(Cl) (LOT: S01C015), Diaion SA10A(Cl) (LOT: T30A023),
Amberlite XADꢀ4 (LOT: 10195020) and Diaion HPA25(Cl) (LOT: U23D040) were
purchased from Alfa Aesar and Dowex 1x2(Cl) (LOT: 427259956) was purchased from Carl
Roth. Amberlite XADꢀ1180N (LOT: BCBD7925V) and LEWATIT VP OC 1064 (LOT:
BCBB2767V) were purchased from Fluka. Amberlite XADꢀ7HP (LOT: 041M0001V) and
Dowex Optipore Lꢀ493 (LOT not given) was purchased from Sigma. Diaion HPꢀ2MG (LOT:
184408F) was purchased from Supelco (see SI).
General Procedure for Biotransformations (small scale)
Lyophilized E. coli cells (60 mg whole cells, containing the corresponding overexpressed
enzyme 2,3ꢀDHBD_Ao), were rehydrated in 900 µL 50 mmol·Lꢀ1 acetate buffer pH 5.2 and
horizontally shaken at 30 °C and 180 rpm for 30 min. A 500 mmol·Lꢀ1 stock solution of the
substrate was prepared in ultrapure water. 100 µL of the substrate stock solution and 300 mg
KHCO3 were added successively to the cell suspension (1 mL final, 50 mmol·Lꢀ1 final
concentration of substrate, 3 mol·Lꢀ1 final concentration of KHCO3) to give a final pH of 8.5.
A blank sample was prepared as described without cells to ensure the absence of a
carboxylation reaction without the catalyst. The vials were tightly sealed and horizontally
shaken at 30 °C and 180 rpm for 24 h.
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After 24 h the reaction was stopped by transferring the whole reaction mixture into 3 mL of
a solution containing 2 mol·Lꢀ1 HCl:MeOH (66:33 v/v). The reaction vessel was additionally
rinsed two times with each 500 µL 2 mol·Lꢀ1 HCl:MeOH (66:33 v/v). The resulting mixture
was centrifuged (5 min, 4000 rpm) and the supernatant was filtered through a 10 kDa
Centrifugal Filter (VWR Modified PES 10K, 500 µl centrifugal filter) to remove residual
proteins (5 min, 14000 rpm). The permeate was diluted in ultrapure water (1:50 total dilution)
and the resulting mixture was analyzed by HPLC (see below).
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For investigations of the reaction system (conversion vs. time), samples for HPLC were
taken after definite time intervals. Therefore 100 µL of the reaction solution was diluted into
400 µL of a solution containing 2 mol·Lꢀ1 HCl:MeOH (66:33 v/v). The mixture was vortexed
to remove remaining bicarbonate and processed as described above (centrifugation, filtration,
1:50 total dilution and analysis by HPLC).
For the evaluation of the biocatalyst concentration the desired mass of whole cell catalyst
(0.5, 1, 2, 5, 10, 20, 30 and 60 mg) was weighed in 1 mL HPLC glass vials and the reaction
conditions were chosen as previously described.
Characteristics and preparation of the anion–exchange and adsorbents beads
The specific characteristics of the resins are shown in Table S1 and Table S2 (see SI). The
anion–exchange resins were used in their native ionic form. Prior to use, all resins were
washed by ultrasonification for 5 min successively with 0.1 mol·Lꢀ1 HCl, ultrapure water and
acetone. Afterwards the resin beads were dried under vacuum (rotary evaporator, 40 °C,
20 mbar).
Four anion exchange resins with strong basicities, according to the supplier’s specifications,
were included in this study. The data in parentheses indicate the type of counterion bound to
the resin in its native form. The total exchange capacity of the resin in mmol equivalents per
gram (meq gꢀ1) is defined as the total number of counterions available for exchange per unit
weight of resin.
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Adsorption experiments
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To evaluate the capability for a downstreamꢀprocessing of the case biocatalytic reaction, the
capacity of the resins with respect to substrate and product were determined. For these
adsorption experiments a 25 mmol·Lꢀ1 test solution of product and substrate in 2.5 mol·Lꢀ1
bicarbonate was prepared. 5 mL of this test solution was then added to 0.1 g, 0.5 g and 1.0 g
of each resin, respectively. The shakingꢀflask assays were performed at 30 °C and 180 rpm
for 30 min. In earlier experiments (data not shown) adsorption equilibrium was determined to
be reached within 30 min. The adsorption of substrate and product was determined by
analysis of the residual concentration in solution before and after adding the adsorbent resins
via HPLC. Therefore, 100 µL of the supernatant was diluted into 900 µL of a solution
containing 2 mol·Lꢀ1 HCl:MeOH (66:33 v/v) to remove remaining bicarbonate and dilution
(2:3 v/v) in ultrapure water. For calculations, the measured concentration of the prepared test
solution was set as 100%.
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Desorption studies experiments
Because of the promising results from the adsorption experiments, desorption experiments
for the anionꢀexchange resin Dowex 1x2 (Cl) were investigated. Therefore, 0.5 g Dowex 1x2
(Cl) was loaded with substrate and product as mentioned above. Different mixtures of
2 mol·Lꢀ1 HCl:acetone were then added to the resin beads for the elution experiments at 30 °C
and 180 rpm for 30 min. Afterwards the concentrations of substrate and product in solution
were determined via HPLC. The supernatant was removed by decantation, and two more
elution steps were performed as described.
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Biocatalytic carboxylation starting with 2.5 g orcinol, including downstream-
processing using anion-exchange resin Dowex 1x2 (Cl)
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For a preparativeꢀscale enzymatic reaction, 12 g lyophilized E. coli cells containing the
corresponding overexpressed enzyme (30 mg cells/mL reaction solution) were rehydrated in
400 mL (50 mmol·Lꢀ1) acetate buffer pH 5.2 and stirred in a tempered and closed 1 L flask at
30 °C and 600 rpm for 30 min. 2.5 g of substrate orcinol and 120 g KHCO3 were added
successively to the cell suspension (50 mmol·Lꢀ1 final concentration of substrate, 3 mol·Lꢀ1
final concentration of KHCO3) to give a final pH of 8.5.
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After 24 h the reaction was checked for maximal conversion by adding a 100 µL sample to
400 µL of a solution containing 2 mol·Lꢀ1 HCl:MeOH (66:33 v/v). The mixture was
centrifuged (5 min, 4000 rpm) and the supernatant filtered through a 10 kDa centrifugal filter
(VWR Modified PES 10K, 500 µl centrifugal filter) to remove residual proteins (5 min,
14000 rpm). The permeate was diluted in ultrapure water (1:50 total dilution) and the
resulting mixture was analyzed by HPLC (see below).
For the removal of DHMBA and orcinol from reaction solution, the cells were first
separated from reaction solution by centrifugation (20 min, 4000 rpm). Then, 40 g of washed
anionꢀexchange resin Dowex 1x2 (Cl) (0.1 g of resin/mL) was added to the supernatant and
stirred for 2 h at 30 °C and 600 rpm. Afterwards, the supernatant was checked for full
adsorption as previously described. The loaded adsorbent beads were separated from the
solution and washed with 20 mL water. The loaded resins were then treated with 400 mL of
2 mol·Lꢀ1 HCl:acetone (20:80 v/v) for 30 min at 30 °C and 600 rpm to recover product and
substrate. Afterwards, the resin beads were filtered off and the permeate evaporated in a
rotary evaporator to remove acetone (40 °C, from 400 mbar at the beginning to 0 mbar in the
end) until white crystals appeared in the remaining aqueous 2 mol·Lꢀ1 HCl solution. After
vacuum filtration, the crystals of DHMBA were dried for 2 h by lyophilisation. This
desorption procedure was performed two more times.
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After carrying out detailed analytics (1HꢀNMR, CꢀNMR, HSQC, methanolic solution of
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received crystals for HPLC and LCꢀMS) no further purification steps were necessary to obtain
pure product in high purity (>99%).
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Analytics
HPLC analysis
Analyses were performed on a HPLC system from Knauer (pump: WellChrom Kꢀ1001,
degasser: Smartline Online Degasser, autosampler: Spark Marathon Basic, software: Clarity
Chrome) equipped with a 4ꢀchannel UV detector (Knauer UV Detector S 2550) and a
reversed phase Phenomenex Kinetex C18 column (100 Å, 150 × 3.00 mm, 2.6 µm, item
number 00Fꢀ4462ꢀY0, column temperature 35 °C). Conversions were determined by using
calibration curves for substrate and product prepared with commercial reference materials. All
compounds were spectrophotometrically detected at 273 nm (orcinol) and 248 nm (DHMBA).
The method was run over 40 min (flow rate 0.2 mL minꢀ1) with water (+0.1% formic acid,
solvent A) and MeOH (+0.1% formic acid, solvent B) as the mobile phase with the following
gradient: 0ꢀ6 min 30% B, 6ꢀ10 min 30ꢀ80% B, 10ꢀ18 min 80% B, 18ꢀ28 min 80ꢀ30% B,
28ꢀ40 min 30% B). Retention times: 8.6 min (orcinol) and 13.2 min (DHMBA).
NMR analysis
1H NMR and 13C NMRꢀspectra were recorded with a Bruker AVANCE (300 MHz) in
DMSOꢀ
d
6. Chemical shifts are reported in parts per million (ppm) relative to the solvent peak,
are given in Hz. Splitting patterns are indicated as follows: s, singlet; d,
coupling constants
J
doublet; t, triplet; m, multiplet; quint, quintet, dt, double triplet; dd, double doublet.
LC-MS analysis
The purity of the target compound (DHMBA) was determined to be >99% by LCꢀMS using
a Thermo Finnigan LTQ Velos Pro LCꢀMS (software: Xcalibur) equipped with an ion trap
detector and reversed phase column (Phenomenex Kinetex Phenyl Hexyl column (100 Å,
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150 × 3.00 mm, 2.6 µm, column temperature 35 °C). The method was run as mentioned
earlier (see “HPLC analysis”).
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3. RESULTS AND DISCUSSION
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The biocatalytic carboxylation of orcinol to DHMBA was initially reported by Wuensch et
al.16 A substrate screening showed that 2,3 DHBD_Ao selectively converts a broad substrate
scope of substrates to the corresponding orthoꢀcarboxylated derivatives. Moderate
conversions were found with a 300ꢀfold excess of bicarbonate, whereas fortunately only
orthoꢀregioisomeric carboxylation products were formed.16,20 However, major limitations are
low substrate loading and the requirement to remove the entire excess bicarbonate via
acidification during downstreamꢀprocessing, followed by column chromatography to purify
the desired product. This study targets to (partly) overcome these limitations.
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3.1 Optimization of the enzymatic carboxylation reaction
Initial experiment targeted the increase of the substrate concentration to facilitate higher
yields within the biocatalytic process. Since the equilibrium conversion is directly linked to
the excess of bicarbonate over orcinol, higher substrate concentrations will inevitably result in
lower equilibrium conversions at a constant bicarbonate concentration of 3 mol·Lꢀ1 (Keq is
approximately 5·10ꢀ4) (Fig. 2, A).
While a substrate concentration of 5 mmol·Lꢀ1 will lead to >80% conversion, higher
substrate concentrations of 10 mmol·Lꢀ1 and 50 mmol·Lꢀ1 result in only 80 and 68%
conversion, respectively. A further increase of orcinol concentration unfortunately leads to a
full inhibition of the catalytic reaction system. Consequently, a substrate concentration of
50 mmol Lꢀ1 was used throughout this study. Catalyst loading was optimized here to facilitate
optimal catalyst usage within the desired process with recombinantly expressed
2,3ꢀDHBD_Ao in E. coli BL21 (DE3) whole cells. As previously reported, a lower substrate
loading of only 5 mmol Lꢀ1 is easily converted by ca. 10 mg whole cell biocatalyst/mL, but for
50 mmol·Lꢀ1 a significant higher catalyst loading of 60 mg whole cell biocatalyst/mL is
required to reach equilibrium conversion (Fig. 2ꢀB and 2ꢀC). This clearly indicates that further
improvements of the catalytic robustness towards higher substrate loadings and higher
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specific activities are required to improve overall process competitiveness. The final
optimized reaction conditions carried out at 30 °C, a substrate concentration of 50 mmol·Lꢀ1
and 3 mol·Lꢀ1 KHCO3 resulted in a conversion of 60% (Fig. 2ꢀD, highlighted with star). It is
important to note that the reaction time course of the upꢀscaled application (see below) differs
from these results since the required sample drawing procedure causes multiple losses of
gaseous carbon dioxide from the reaction mixture. This results in a reduced excess of
bicarbonate/CO2, which induces lower equilibrium conversions. Hence, a closed vessel
reaction setꢀup was preferred throughout this study, especially at a larger scale.
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5 mmol ⋅ Lꢀ1 of substrate
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amount of biocatalyst / mg ⋅ mL-1
C
D
50 mmol ⋅ Lꢀ1 of substrate
100
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10
0
0.5
1
2
5
10 20 30 60
0
4
8
12 16 20 24
amount of biocatalyst / mg ⋅ mL-1
reaction time / h
Fig. 2. A) conversion of different orcinol concentrations with a fixed bicarbonate
concentration of 3 mol·Lꢀ1 with 30 mg·Lꢀ1 biocatalyst at 30 °C; B) required catalyst loading to
reach equilibrium conversion at 5 mmol·Lꢀ1 orcinol and 3 mol·Lꢀ1 bicarbonate; C) required
catalyst loading to reach equilibrium conversion at 50 mmol·Lꢀ1 substrate and 3 mol·Lꢀ1
bicarbonate; D) time course and final reactant concentration at optimized conditions; reaction
conditions: 50 mmol·Lꢀ1 NaOAc buffer solution, 3 mol·Lꢀ1 KHCO3,
T
= 30 °C,
U
= 180 rpm,
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∑t
= 24 h, final pH 8.5. All experiments were carried out in triplicate and the calculated
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standard deviations are shown.
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3.2 Selection of adsorbents resin
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Classical downstream concepts for carboxylic acids, e.g. from fermentation processes,
include mainly the use of adsorption,21 extraction22 or crystallization.23 Unfortunately, to date,
no effective recovery method of aromatic hydroxy acids for an enzymatic carboxylation
reaction have been reported in scientific literature. This mainly involves the significant excess
of 3 mol·Lꢀ1 bicarbonate, which is required to push the reaction equilibrium to favor the
product side. This excess is afterwards removed via acidification, resulting in a full loss of
bicarbonate and thus lowering atom efficiency of the overall reaction system.
The main objective of the first downstreamꢀprocessing step is to remove both reactants
from the bicarbonate solution, which might thereafter be recycled. Different commercially
available anion–exchange and nonꢀionic adsorbents resins were screened for their ability to
remove both product (DHMBA) and remaining substrate (orcinol) from a decarboxylase
reaction solution in the presence of excess bicarbonate, as mentioned above. Therefore, test
solutions containing 25 mmol·Lꢀ1 of product and substrate in 2.5 mol·Lꢀ1 KHCO3 were
prepared to simulate the reaction system at a conversion of 50 %. Furthermore, three different
loading capacities (0.1, 0.5 and 1.0 g per 5 mL test solution, respectively) were evaluated to
estimate the required loading of the adsorber materials. The residual concentration in solution
was measured via HPLC and the results are shown in Table 1.
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Table 1. Adsorption of DHMBA and orcinol onto ten different adsorbers (anionic and nonꢀ
inonic form) at 30 °C and 180 rpm after 30 min from test solution containing 25 mmol·Lꢀ1
either of product and substrate in 2.5 mol·Lꢀ1 KHCO3.
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applied adsorber
DHMBA adsorption/%
orcinol adsorption/%
mL-1
0.2 g
mL-1
—
0.02 g
·
mL-1 0.1 g
·
mL-1 0.2 g
·
mL-1
0.02 g
mL-1
·
0.1 g
—
a) anion exchanger resins
Dowex 1x2 (Cl)
93
77
83
74
>99
99
>99
>99
>99
>99
74
51
52
46
>99
96
>99
>99
>99
>99
Diaion PA312 (Cl)
Diaion HPA-25 (Cl)
Diaion SA10A (Cl)
b) non-ionic adsorbent
Dowex Optipore L493
Lewatit VP OC
98
98
99
96
48
20
33
42
1
97
44
90
93
7
>99
61
97
98
16
13
57
21
44
47
3
98
55
93
94
19
8
>99
72
Diaion HP-2MG
>99
>99
29
Amberlite XAD-7HP
Amberlite XAD-4
Amberlite XAD-1180N
2
4
1
14
In general, ion exchange resins exhibit a much stronger affinity towards orcinol and
DHMBA in comparison to nonꢀionic adsorber materials. The affinity of the ion exchange
resins ordered in decreasing order of affinity was: Dowex 1x2 (Cl) > Diaion HPAꢀ25 (Cl) >
Diaion PA312 (Cl) > Diaion SA10A (Cl), while the nonꢀionic adsorbers were ordered as
Dowex Optipore L493 > Amberlite XADꢀ7HP > Diaion HPꢀ2MG > Lewatit VP OC >
Amberlite XADꢀ4 > Amberlite XADꢀ1180N. The highest adsorption affinity towards orcinol
and DHMBA was found for Dowex 1x2 (Cl), which requires only 0.1 g adsorber per mL
reaction solution to fully remove both main reactants. Thus, Dowex 1x2 (Cl) was
subsequently used throughout the study.
In addition, all anion–exchange resins show a slightly higher adsorption affinity towards the
product DHMBA in comparison to the substrate orcinol. The highest difference between the
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adsorption of DHMBA and orcinol can be achieved with the Diaion HPAꢀ25 (Cl) resin (31%),
which might be used in future application within a (partial) in situ product removalꢀstrategy
(ISPR). Here the shown difference in adsorption is based on the presence of the respective
microspecies of both reactants. At pH 8.4, which originates from 3 mol·Lꢀ1 KHCO3
(highlighted as dashed line in Fig. 3), the carboxylic group of DHMBA is fully deprotonated
forming the corresponding monoanion of DHMBA. In contrast, the phenolic hydroxyl group
of orcinol is only partly deprotonated and thus the majority of the substrate is present in its
neutral form.
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A Orcinol
B DHMBA
100
100
#2A
#1A
#3A
80
60
40
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0
80
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#2B
#1B
20
0
#3B
#4B
0
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10 12 14
0
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8
10 12 14
pH
pH
Microspecies #1A
Microspecies #2A
Microspecies #1B
Microspecies #2B
Microspecies #3A
Microspecies #3B
Microspecies #4B
Fig. 3. Undissociated fraction (1A, 1B), monodissociated fractions (2A, 2B), double
dissociated fraction (3A, 3B) and triple dissociated fraction (4B) of the total of orcinol [A]
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and DHMBA [B] species. Diagrams and pKs values calculated with “chemicalize”
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(www.chemicalize.com).
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Consequently, DHMBA adsorbs more strongly via ionic interactions towards ion exchange
resins than its counterpart orcinol. These ionic interactions overlap with nonꢀspecific
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hydrophobic adsorption of both reactants towards the polystyrene (ST)/pꢀdivinylbenzene
(DVB)ꢀpolymer backbone of the ion exchange resin, which in combination result in the
observed affinities (Fig. 4).
Fig. 4. General presentation of ST (blue)/DVB (red) based anion exchange resin structure
with trimethylbenzylammonium functional groups.
In comparison, the investigated nonꢀionic adsorber materials facilitate only hydrophobic
interactions and thus show no significant differences in adsorption between orcinol and
DHMBA.
3.3 Desorption from Dowex 1x2 (Cl) and Final Precipitation of DHMBA
After loading, the adsorbent including both reactants were removed from the reaction
mixture to facilitate its separation from the remaining concentrated bicarbonate solution.
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After loading experiments, elution experiments were performed with different organic
solvents and aqueous solutions. Optimal desorption from the loaded adsorbent was found with
a solvent mixture consisting of 2 mol·Lꢀ1 HCl and acetone, which allows a direct combination
with the final precipitation step (see below). Different mixtures were investigated, and the
results are presented in Fig. 5.
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Fig. 5. Desorption of orcinol and DHMBA from loaded anionꢀexchange resin Dowex 1x2 (Cl)
at different mixtures of 2 mol·Lꢀ1 HCl/acetone at 30 °C after 30 min for each desorption step.
Arrows are shown only as a guide (filled arrow: decrease of orcinol desorption with
decreasing acetone content; open arrow: increase of DHMBA desorption with decreasing
acetone content).
By increasing the ratio of 2 mol·Lꢀ1 HCl from 10% to 40% (v/v) the initial desorption of
DHMBA increases successively from 40% to a maximum of 50% (open arrow) with a
simultaneous reduction of orcinol elution from 86% to 72% (filled arrow). Eventually a
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solvent mixture of 2 mol·Lꢀ1 HCl:acetone (20:80 v/v) was sufficient with at least three
desorption steps to reach a recovery rate of ≥90% for both reactants.
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After filtration of the depleted adsorbent Dowex 1x2 (Cl) and evaporation of acetone, the
free acid DHMBA directly precipitates from the remaining aqueous HClꢀsolution. The
depleted adsorbent and the remaining orcinol (in solution) can then be reꢀused. After a single
filtration, pure DHMBA was yielded. A preparativeꢀscale enzymatic carboxylation of orcinol,
as a concept study, yielded 878 mg of DHMBA with >99% purity in crystalline form without
any further purification steps, e.g. by column chromatography or reꢀcrystallization. In contrast
to classical downstreamꢀprocessing approaches of biocatalytic carboxylation reactions, a total
elimination of the excess of bicarbonate is not required.
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SUMMARY AND CONCLUSION
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In this contribution the process development of the decarboxylaseꢀcatalyzed synthesis of
2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid (DHMBA) by an adsorbentꢀbased downstreamꢀ
processing approach is presented. The biocatalytic reaction was optimized and a variety of
adsorbent resins were investigated for the effective removal of both product and substrate
from the bicarbonateꢀcontaining reaction solution. Here the strongly basic anion exchange
resin Dowex 1x2 (Cl) was found to be the most effective adsorbent and was subsequently
used within this study. After elution studies, the resin was successfully used for the
downstreamꢀprocessing of a scaled up reaction (from 1 to 400 mL) to produce highly pure
(>99%) DHMBA without further purification steps.
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The developed process concept herein enables the synthetic use of the biocatalytic
carboxylation reaction without the full removal of excess bicarbonate during downstreamꢀ
processing. Unreacted substrate and the depleted resins can be obtained from the reaction
mixture and reꢀused. This case study highlights the synthetic potential of biocatalytic
carboxylation reactions and may serve as a model for other carboxylation biotransformations,
especially at a larger scale.
AUTHOR INFORMATION
* Jan von Langermann: jan.langermann@uniꢀrostock.de
University of Rostock, Institute of Chemistry, AlbertꢀEinsteinꢀStr. 3a, 18059 Rostock,
Germany
# Udo Kragl: udo.kragl@uniꢀrostock.de
Funding Sources
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Financial support is gratefully acknowledged by the DFG (Deutsche
Forschungsgemeinschaft), grant number: 252186816 and FWF (Fonds zur Förderung der
wissenschaftlichen Forschung), grant number: 1637 (ERA; ERA Chemistry Open Call 6;
ERA Chemistry Open Initiative).
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ACKNOWLEDGMENTS
The authors thank the entire research group of Prof. Dr. Hubert Bahl (Department of
Microbiology, University of Rostock) for their ongoing support and useful discussions. The
authors also thank Marek Biermann and Lukas Friederici for their assistance in the
experimental work.
ABBREVIATIONS
2,3ꢀDHBD_Ao, (de)carboxylase from Aspergillus oryzae; DHMBA, 2,6ꢀdihydroxyꢀ4ꢀ
methylbenzoic acid; DSP, downstreamꢀprocessing; PSꢀDVB, Polystyrene/pꢀDivinylbenzene
SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS Publications website at
DOI: XXX and contains a summary of characteristics of the investigated resins and NMRꢀ
data of the obtained 2,6ꢀdihydroxyꢀ4ꢀmethylbenzoic acid.
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