Letter pubs.acs.org/acscatalysis
An Unprecedented Effective Enzymatic Carboxylation of Phenols Jie Ren, Peiyuan Yao, Shanshan Yu, Wenyue Dong, Qijia Chen, Jinhui Feng, Qiaqing Wu, and Dunming Zhu*
ACS Catal. 2016.6:564-567. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 01/29/19. For personal use only.
National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Research Center for Biocatalytic Technology Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, China ABSTRACT: It is well-known that the equilibrium of the enzymatic Kolbe−Schmitt reaction is unfavorable for the carboxylation direction. A new method was developed to push the reaction equilibrium toward the carboxylation of resorcinol and catechol by adding quaternary ammonium salts into the reaction system. The yields of the carboxylation products were increased up to 97% from less than 40%. The precipitation capacity of the quaternary ammonium salts of the carboxylic acid products is the driving force for the reaction equilibrium shift. Therefore, effective enzymatic carboxylation of phenols has been achieved for the first time. KEYWORDS: enzymatic carboxylation, reaction equilibrium, quaternary ammonium salts, carboxylase, decarboxylase, Kolbe−Schmitt reaction
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toward the carboxylation of the phenols by changing the reaction conditions. Herein, we report a new strategy to drive the equilibrium of decarboxylase-catalyzed reaction toward the formation of carboxylation products by adding quaternary ammonium salts into the reaction system as the precipitant for the obtained salicylic acid derivatives, as shown in Scheme 1.
he Kolbe−Schmitt process is well-known in organic synthesis because the carboxylation of phenols provides a unique approach to produce aromatic acid by employing CO2 as C1 source. However, the reaction suffers from unsatisfactory regioselectivity toward the ortho- or para- carboxylation and requires harsh reaction conditions such as high temperature and CO2 pressure.1 Some attempts have been made to overcome these disadvantages by employing metal catalysis,2 microwave-assisted reactions,3 and ionic liquids as solvent.4 Recently, the enzymatic Kolbe−Schmitt reaction has been investigated by employing reversible nonoxidative decarboxylases in biodegradation pathways,5 such as 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3DHBD_Ao),6 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. (2,6-DHBD_Rs),7 and salicylic acid decarboxylase from Trichosporon moniliiforme (SAD_Tm).8 These enzymes catalyze the decarboxylation of benzoic acid derivatives and the reverse carboxylation reaction of phenols to the corresponding salicylic acid derivatives in the presence of bicarbonate as CO2 source. The enzymatic carboxylation reactions not only avoided the harsh reaction conditions but also showed exclusive regioselectivity in the ortho-position and broad substrate spectrum.9 However, these enzymatic reactions are reversible. The enzyme kinetics and reaction thermodynamics indicated that the reaction equilibrium was unfavorable for the carboxylation direction10 but more preferred to the decarboxylation direction. As such, the reported carboxylation conversions were limited to 40% for most of the decarboxylases and phenols. The reaction conditions of resorcinol carboxylation catalyzed by 2,6-DHBD_Rs were optimized by varying pH, temperature, substrate concentration, and the use of organic cosolvents and ionic liquids; with these modifications, the best conversion of 50% has been achieved.11 These results indicate that it is difficult to drive the reaction equilibrium © 2015 American Chemical Society
Scheme 1. Equilibrium Shift to Carboxylation Direction by Adding Quaternary Ammonium Salts As Precipitant
It has been reported that 2,6-dihydroxybenzoic acid and tetrabutylammonium chloride could form the salt, which precipitated in alkaline solution.12 According to the Le Chatelier’s principle, we envisaged that in the presence of quaternary ammonium cation in the reaction system, the carboxylation product could form ammonium salt, which would precipitate continually, thus driving the reaction equilibrium in favor of the carboxylation direction. In order to prove this concept, eight quaternary ammonium salts were tested as the precipitant of the product acids in the enzymatic carboxylation. These salts included tetrabutylammonium bromide (N1), noctyltrimethylammonium chloride (N2), dodecyltrimethylReceived: November 10, 2015 Revised: December 15, 2015 Published: December 17, 2015 564
DOI: 10.1021/acscatal.5b02529 ACS Catal. 2016, 6, 564−567
Letter
ACS Catalysis
Table 1. Initial Reaction Rates and Product Yields of Resorcinol Carboxylation after Adding Quaternary Ammonium Salts and Tetrabutylphosphonium Bromidea amount of added quaternary ammonium salts (20 mM)
entry
quaternary ammonium salts
initial reaction rate (mM h−1)
yield of 2,6dihydroxybenzoic acid (%)
1 2 3 4 5 6 7 8 9 10
controlb N1 N2 N3 N4 N5 N6 N7 N8 N9
7.2 7.9 7.7 5.8 5.5 5.3 8.6 1.4 5.0 8.6
37 91 41 72 88 82 37 90 89 83
a b
amount of added quaternary ammonium salts (50 mM)
yield of 2,4dihydroxybenzoic acid (%)
yield of 2,6dihydroxybenzoic acid (%)
yield of 2,4dihydroxybenzoic acid (%)
1.7 0.5 1.8 1.0 0.3 0.5 1.6 0.2 0.2 0.5
37 97 79 91 95 95 46 52 59 87
1.7 200 >200 45.5 37.4 >200
154.6 >200 >200 >200 >200 >200 43.9 49.0 >200
>200 >200 >200 >200 >200 89.5 0.5 0.5 >200
73.0 >200 >200 >200 >200 >200 24.0 21.2 >200
a
The residual concentration of the carboxylic acid or phenol was determined by HPLC analysis. bNo precipitate was formed under the test conditions, the apparent solubility product constant should thus be greater than 2 × 10−4 M2. 566
DOI: 10.1021/acscatal.5b02529 ACS Catal. 2016, 6, 564−567
Letter
ACS Catalysis
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solution and rehydrated for 30 min. The substrate (resorcinol or catechol, 10 mM) and the quaternary ammonium salt or tetrabutylphosphonium bromide (20 mM) were added into the resulting mixture. The mixture was shaken at 30 °C for 10 min, and the initial reaction rate was measured by HPLC analysis. For the determination of the product yield, the reaction mixture was shaken at 30 °C for 24 h (or 48 h for catechol carboxylation). For HPLC analysis, the reaction mixture was added dropwise into a solution containing 2 mL of 2 M HCl and 2 mL of methanol. The resulting mixture was centrifuged at 21 000g for 2 min, and the supernatant was analyzed by HPLC, which was performed on an Agilent 1200 system with an Eclipse XDB-C18 column (4.6 × 150 mm). A mixture of acetonitrile (15%) and 0.3% trifluoroacetic acid solution in water (85%) were used as mobile phase. Preparative Scale Carboxylation of Resorcinol in the Presence of Tetrabutylammonium Bromide. Resorcinol (220 mg), tetrabutylammonium bromide (800 mg), KHCO3 (3 g), and lyophilized E. coli cells contained decarboxylase 2,6DHBD_Rs (400 mg) were mixed in 10 mL of distilled water, and the resulting mixture was shaken at 30 °C for 24 h. More lyophilized E. coli cells (400 mg) was added and the mixture was shaken for another 24 h at 30 °C. HPLC analysis indicated that the yield reached 99%. The precipitate was filtrated and washed with 20 mL of distilled water, dried at 60 °C to give the salt as white solid (660 mg, 90% yield). The obtained white solid (600 mg) was dissolved in 3 mL of hydrochloric acid. After standing for 1 h, 2,6-dihydroxybenzoic acid was precipitated and separated by filtration. The precipitate was washed with 8 mL of distilled water and dried under vacuum to give 2,6-dihydroxybenzoic acid as dark pink solid (170 mg, 72% yield). The quaternary ammonium salt in mother liquor was recovered by extracting with chloroform. Measurement of Apparent Solubility Product Constants. Substrate phenol or product carboxylic acid was mixed with quaternary ammonium salts or tetrabutylphosphonium bromide in 1 mL of saturated KHCO3 solution with their final concentrations being 10 mM and 20 mM, respectively. The mixture was stirred at 30 °C for 1 h. After filtration, the concentration of phenol or product carboxylic acid in the supernatant was measured by HPLC analysis. The apparent solubility product constants were calculated and the results are summarized in Table 3.
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AUTHOR INFORMATION
Corresponding Author
*E-mail for D.Z.:
[email protected]. Fax: +86 22 84861996. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was financially supported by the National Natural Science Foundation of China (Grant No. 21472232)
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REFERENCES
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DOI: 10.1021/acscatal.5b02529 ACS Catal. 2016, 6, 564−567
