Appl Biochem Biotechnol DOI 10.1007/s12010-014-0840-3
Naturally Occurring Alkaline Amino Acids Function as Efficient Catalysts on Knoevenagel Condensation at Physiological pH: A Mechanistic Elucidation Weina Li & Sergey Fedosov & Tianwei Tan & Xuebing Xu & Zheng Guo
Received: 21 December 2013 / Accepted: 27 February 2014 # Springer Science+Business Media New York 2014
Abstract To maintain biological functions, thousands of different reactions take place in human body at physiological pH (7.0) and mild conditions, which is associated with health and disease. Therefore, to examine the catalytic function of the intrinsically occurring molecules, such as amino acids at neutral pH, is of fundamental interests. Natural basic α-amino acid of L-lysine, L-arginine, and L-histidine neutralized to physiological pH as salts were investigated for their ability to catalyze Knoevenagel condensation of benzaldehyde and ethyl cyanoacetate. Compared with their free base forms, although neutralized alkaline amino acid salts reduced the catalytic activity markedly, they were still capable to perform an efficient catalysis at physiological pH as porcine pancreatic lipase (PPL), one of the best enzymes that catalyze Knoevenagel condensation. In agreement with the fact that the three basic amino acids were well neutralized, stronger basic amino acid Arg and Lys showed more obvious variation in NH bend peak from the FTIR spectroscopy study. Study of ethanol/water system and quantitative kinetic analysis suggested that the microenvironment in the vicinity of amino acid salts and protonability/deprotonability of the amine moiety may determine their catalytic activity and mechanism. The kinetic study of best approximation suggested that the random binding might be the most probable catalytic mechanism for the neutralized alkaline amino acid salt-catalyzed Knoevenagel condensation. Keywords Knoevenagel condensation . Amino acid . Lipase . Neutralized alkaline amino acid salt . Physiological pH
Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0840-3) contains supplementary material, which is available to authorized users.
W. Li : S. Fedosov : X. Xu : Z. Guo (*) Department of Engineering, Aarhus University, Aarhus, Denmark e-mail: [email protected]
W. Li : T. Tan (*) College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Knoevenagel condensation, a modified aldol condensation for classic C–C bond formation, occurs between aldehydes or ketones and the compounds containing active methylene [1]. As many classic reactions, Knoevenagel condensation has been studied either for an efficient catalysis [2–9] or in the perspective of green chemistry [10–16]. As listed in Table 1, this reaction has been intensively studied under a variety of conditions, solvents, and catalysts in recent years. As cheap and environmentally benign compounds, α-amino acids have been widely used as a catalyst for Knoevenagel condensations, among which naturally occurring secondary amino acid L-proline and primary amino acids L-arginine, L-lysine, and L-histidine displayed good activity [12–16]. Two possible mechanisms have been proposed for this reaction [16]. In the Hann–Lapworth hypothesis an intermediate α-hydroxydicarbonyl compound is assumed, followed by dehydration to the alkene (Scheme 1, Path A). The postulated mechanism would involve catalysis of a weak base and a weak acid as listed in Table 1. In the other mechanism shown in Scheme 1, Path B, proline in its zwitterionic form reacts with benzaldehyde (A0), forming corresponding iminium intermediate. Then reacted with B0 if the intermediary diastereomeric iminium ions or Mannich products (if generated reversibly) to give the Knoevenagel product. However, a decisive kinetic support for the mechanism and a quantitative characterization has scarcely been obtained [2–4], which will be one of the focuses in this work. Moreover, all the amino acids used as a catalyst in Knoevenagel condensation in the studies reported so far are all in free base form [12–16]. While to investigate amino acid-mediated Knoevenagel condensation reaction at physiological pH 7.4 may be of scientific value for a better understanding of the possible Knoevenagel condensation reaction in human body [17], which is related to human nutrition and health [18]. As known, aldehydes and malonic acid, etc., are the major products of lipid secondary oxidation, which may generate cytotoxicity and genotoxicity through interaction between the products and interactions with other small or macromolecules in human body including Knoevenagel condensation reactions [19, 20]. Therefore, in this study, we (1) prepared neutralized alkaline amino acid HCl salts (L-lysine, Larginine, L-histidine) at a neutral pH 7.0 (close to the physiological pH); (2) investigated the catalytic behaviors of these neutralized amino acid salts for Knoevengael condensation, in terms of reaction conditions and substrate spectrum; (3) performed a quantitative kinetic study to elucidate the reaction mechanism; (4) compared with the reactions, under other identical conditions, catalyzed by the amino acids as a free base and porcine pancreas lipase (PPL), which is one of the most efficient lipases observed for Knoevengael condensation [11]. It turned out that, compared with free base amino acids, the neutralized salts showed a different activity response to the change of water concentration, and a decreased activity relative to its free base counterpart, but still was able to achieve a considerable good catalytic activity as obtained by one of the most efficient lipases, PPL.
Experimental Commercial reagents and enzymes were all obtained from Sigma-Aldrich. Solvents for kinetics study were dried by molecular sieves. Protein concentrations were determined by Bradford method using bovine serum albumin as a standard. The protein content for porcine pancreatic lipase is 12.6 % (wt.%).
Room temperature
Room temperature
DMSO Water
DMSO DMSO Ethanol
Benzaldehyde and malonic ester derivative[ethyl cyanoacetate (ECA)]
Aromatic aldehydes with malononitrile or ethyl cyanoacetate
Benzaldehyde + diethyl malonate
Benzaldehyde and diethyl malonate
Benzaldehyde + malononitrile
2-Naphthaldehyde with Meldrum’s acid
Aromatic aldehydes with various active methylene groups
Benzaldehyde + methyl cyanoacetate aromatic
α,β-Unsaturated aldehydes and 1,3-dicarbonyl compounds
Aldehyde with an activated CH acid such as ethyl cyanoacetate, malononitrile, acetyl acetone, or ethyl acetoacetate
Dimethylmalonate and a variety of aldehydes
Hydratropaldehyde + diethylmalonate
Indole-3-carboxyaldehydes + ethyl cyanoacetate
CsNaX zeolites grafted with amino groups
Cetyltrimethyl ammonium bromide
Methylation of the nitrogen-substituted mesoporous silica SBA-15
Amine functionalized K10 montmorillonite, recycled
MCM-41/Schiff base, recycled
Cationic coordination cage
Biodegradable choline chloride, recycled
Lipase from porcine pancreas
Lys
Arg, His
Pro
√ stands for the according experiment has run to verify the result
Room temperature
DMSO/ethanol
n-Ethylcarbazolecarboxaldehyde + p-phenylene-diacetonitrile
Water
Water
Water
No solvent
Ethanol
Water
No solvent
Methano, THF/MeOH mixtures
Room temperature
Room temperature
37 °C/40 °C
25–30 °C
Room temperature
Room temperature
Room temperature
353 K
Room temperature
363 K
65 °C
99.5 °C
Bu4NOH
Kerosene solution
Diethyl malonate and benzaldehyde or ring-substituted benzaldehydes
Temperature
Piperidine
Solvent
Reaction
Catalyst/recycled
Table 1 Catalysts used for Knoevenagel condensation
[4]
Path B √ / Path B Path B
/ √ √ √
/ 98 %/30 min
Path A Path A,B
/ √
/
√ >66 %/16 h
Path A
√ 77–97 %/5–12 h
/
√
/
Path A
√
√
Path A
√
[16]
[15]
[14]
[13]
[12]
[11]
[10]
[9]
[8]
[7]
[6]
[5]
[3]
Path B √
/
[2]
Path B√
√
Reference
Possible mechanisms
Substrate scope study
33–98 %/1–24 h
85/12 h/75/10 h
>90 %/
Naturally Occurring Alkaline Amino Acids Function as Efficient Catalysts on Knoevenagel Condensation at Physiological pH: A Mechanistic Elucidation Weina Li & Sergey Fedosov & Tianwei Tan & Xuebing Xu & Zheng Guo
Received: 21 December 2013 / Accepted: 27 February 2014 # Springer Science+Business Media New York 2014
Abstract To maintain biological functions, thousands of different reactions take place in human body at physiological pH (7.0) and mild conditions, which is associated with health and disease. Therefore, to examine the catalytic function of the intrinsically occurring molecules, such as amino acids at neutral pH, is of fundamental interests. Natural basic α-amino acid of L-lysine, L-arginine, and L-histidine neutralized to physiological pH as salts were investigated for their ability to catalyze Knoevenagel condensation of benzaldehyde and ethyl cyanoacetate. Compared with their free base forms, although neutralized alkaline amino acid salts reduced the catalytic activity markedly, they were still capable to perform an efficient catalysis at physiological pH as porcine pancreatic lipase (PPL), one of the best enzymes that catalyze Knoevenagel condensation. In agreement with the fact that the three basic amino acids were well neutralized, stronger basic amino acid Arg and Lys showed more obvious variation in NH bend peak from the FTIR spectroscopy study. Study of ethanol/water system and quantitative kinetic analysis suggested that the microenvironment in the vicinity of amino acid salts and protonability/deprotonability of the amine moiety may determine their catalytic activity and mechanism. The kinetic study of best approximation suggested that the random binding might be the most probable catalytic mechanism for the neutralized alkaline amino acid salt-catalyzed Knoevenagel condensation. Keywords Knoevenagel condensation . Amino acid . Lipase . Neutralized alkaline amino acid salt . Physiological pH
Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0840-3) contains supplementary material, which is available to authorized users.
W. Li : S. Fedosov : X. Xu : Z. Guo (*) Department of Engineering, Aarhus University, Aarhus, Denmark e-mail: [email protected]
W. Li : T. Tan (*) College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Knoevenagel condensation, a modified aldol condensation for classic C–C bond formation, occurs between aldehydes or ketones and the compounds containing active methylene [1]. As many classic reactions, Knoevenagel condensation has been studied either for an efficient catalysis [2–9] or in the perspective of green chemistry [10–16]. As listed in Table 1, this reaction has been intensively studied under a variety of conditions, solvents, and catalysts in recent years. As cheap and environmentally benign compounds, α-amino acids have been widely used as a catalyst for Knoevenagel condensations, among which naturally occurring secondary amino acid L-proline and primary amino acids L-arginine, L-lysine, and L-histidine displayed good activity [12–16]. Two possible mechanisms have been proposed for this reaction [16]. In the Hann–Lapworth hypothesis an intermediate α-hydroxydicarbonyl compound is assumed, followed by dehydration to the alkene (Scheme 1, Path A). The postulated mechanism would involve catalysis of a weak base and a weak acid as listed in Table 1. In the other mechanism shown in Scheme 1, Path B, proline in its zwitterionic form reacts with benzaldehyde (A0), forming corresponding iminium intermediate. Then reacted with B0 if the intermediary diastereomeric iminium ions or Mannich products (if generated reversibly) to give the Knoevenagel product. However, a decisive kinetic support for the mechanism and a quantitative characterization has scarcely been obtained [2–4], which will be one of the focuses in this work. Moreover, all the amino acids used as a catalyst in Knoevenagel condensation in the studies reported so far are all in free base form [12–16]. While to investigate amino acid-mediated Knoevenagel condensation reaction at physiological pH 7.4 may be of scientific value for a better understanding of the possible Knoevenagel condensation reaction in human body [17], which is related to human nutrition and health [18]. As known, aldehydes and malonic acid, etc., are the major products of lipid secondary oxidation, which may generate cytotoxicity and genotoxicity through interaction between the products and interactions with other small or macromolecules in human body including Knoevenagel condensation reactions [19, 20]. Therefore, in this study, we (1) prepared neutralized alkaline amino acid HCl salts (L-lysine, Larginine, L-histidine) at a neutral pH 7.0 (close to the physiological pH); (2) investigated the catalytic behaviors of these neutralized amino acid salts for Knoevengael condensation, in terms of reaction conditions and substrate spectrum; (3) performed a quantitative kinetic study to elucidate the reaction mechanism; (4) compared with the reactions, under other identical conditions, catalyzed by the amino acids as a free base and porcine pancreas lipase (PPL), which is one of the most efficient lipases observed for Knoevengael condensation [11]. It turned out that, compared with free base amino acids, the neutralized salts showed a different activity response to the change of water concentration, and a decreased activity relative to its free base counterpart, but still was able to achieve a considerable good catalytic activity as obtained by one of the most efficient lipases, PPL.
Experimental Commercial reagents and enzymes were all obtained from Sigma-Aldrich. Solvents for kinetics study were dried by molecular sieves. Protein concentrations were determined by Bradford method using bovine serum albumin as a standard. The protein content for porcine pancreatic lipase is 12.6 % (wt.%).
Room temperature
Room temperature
DMSO Water
DMSO DMSO Ethanol
Benzaldehyde and malonic ester derivative[ethyl cyanoacetate (ECA)]
Aromatic aldehydes with malononitrile or ethyl cyanoacetate
Benzaldehyde + diethyl malonate
Benzaldehyde and diethyl malonate
Benzaldehyde + malononitrile
2-Naphthaldehyde with Meldrum’s acid
Aromatic aldehydes with various active methylene groups
Benzaldehyde + methyl cyanoacetate aromatic
α,β-Unsaturated aldehydes and 1,3-dicarbonyl compounds
Aldehyde with an activated CH acid such as ethyl cyanoacetate, malononitrile, acetyl acetone, or ethyl acetoacetate
Dimethylmalonate and a variety of aldehydes
Hydratropaldehyde + diethylmalonate
Indole-3-carboxyaldehydes + ethyl cyanoacetate
CsNaX zeolites grafted with amino groups
Cetyltrimethyl ammonium bromide
Methylation of the nitrogen-substituted mesoporous silica SBA-15
Amine functionalized K10 montmorillonite, recycled
MCM-41/Schiff base, recycled
Cationic coordination cage
Biodegradable choline chloride, recycled
Lipase from porcine pancreas
Lys
Arg, His
Pro
√ stands for the according experiment has run to verify the result
Room temperature
DMSO/ethanol
n-Ethylcarbazolecarboxaldehyde + p-phenylene-diacetonitrile
Water
Water
Water
No solvent
Ethanol
Water
No solvent
Methano, THF/MeOH mixtures
Room temperature
Room temperature
37 °C/40 °C
25–30 °C
Room temperature
Room temperature
Room temperature
353 K
Room temperature
363 K
65 °C
99.5 °C
Bu4NOH
Kerosene solution
Diethyl malonate and benzaldehyde or ring-substituted benzaldehydes
Temperature
Piperidine
Solvent
Reaction
Catalyst/recycled
Table 1 Catalysts used for Knoevenagel condensation
[4]
Path B √ / Path B Path B
/ √ √ √
/ 98 %/30 min
Path A Path A,B
/ √
/
√ >66 %/16 h
Path A
√ 77–97 %/5–12 h
/
√
/
Path A
√
√
Path A
√
[16]
[15]
[14]
[13]
[12]
[11]
[10]
[9]
[8]
[7]
[6]
[5]
[3]
Path B √
/
[2]
Path B√
√
Reference
Possible mechanisms
Substrate scope study
33–98 %/1–24 h
85/12 h/75/10 h
>90 %/
