“…Resynthesis and Hydrolytic Activity. As was mentioned above, fluorescence of beads containing hits with lysine residues probably originated from aminolysis of the subtrate, 31 which is a noncatalytic process. Since we were interested in catalysis, hits containing lysine residues were not considered for resynthesis.…”
Section: Resultsmentioning
confidence: 90%
“…Instead, the lysine -amine reacted as a nucleophile, even though the pH was far below the pK a of the -amine (pK a ∼10.5), 30 resulting in aminolysis of the substrate. 31 However, increasing the substrate concentration to 100-800 µM revealed the preference of histidine over lysine residues in the basic arm of the synthetic receptors (Figure 3, entries 8-10). Third, the hits also showed that histidine residues seem to be more important for hydrolysis than serine residues.…”
In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.
“…Resynthesis and Hydrolytic Activity. As was mentioned above, fluorescence of beads containing hits with lysine residues probably originated from aminolysis of the subtrate, 31 which is a noncatalytic process. Since we were interested in catalysis, hits containing lysine residues were not considered for resynthesis.…”
Section: Resultsmentioning
confidence: 90%
“…Instead, the lysine -amine reacted as a nucleophile, even though the pH was far below the pK a of the -amine (pK a ∼10.5), 30 resulting in aminolysis of the substrate. 31 However, increasing the substrate concentration to 100-800 µM revealed the preference of histidine over lysine residues in the basic arm of the synthetic receptors (Figure 3, entries 8-10). Third, the hits also showed that histidine residues seem to be more important for hydrolysis than serine residues.…”
In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.
“…Catalysis by phase-transfer agents (crown ethers or glymes) of aminolysis reactions of carboxylic esters is a well-studied process. − The generally accepted catalytic mechanism in aprotic solvents is shown in Scheme . 1 …”
Butylaminolysis of p-nitrophenyl acetate in chlorobenzene in the presence of different kinds of phase-transfer catalysts (crown ethers and glymes) supports the existence of a reaction pathway exhibiting a first-order dependence on the concentration of the phase transfer catalyst and a second-order dependence on the concentration of butylamine. This novel reaction pathway must be included in the mechanism traditionally accepted for the catalysis by phase-transfer agents of aminolysis reactions in aprotic solvents.
“…Catalysis by phase transfer agents (crown ethers or glymes) of aminolysis reactions of carboxylic esters is a well studied process. [1][2][3][4][5][6][7][8] The generally accepted catalytic mechanism in aprotic solvents is shown in Scheme 1. Nucleophilic attack of n-butylamine on the ester generates a tetrahedral intermediate, T ¡ .…”
The mechanism of glyme catalyzed ester aminolysis in chlorobenzene should be modified by including a new reaction pathway that shows a first-order dependence on the concentration of the phase transfer catalyst and a second-order dependence on butylamine.
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