TAC-Scaffolded Tripeptides as Artificial Hydrolytic Receptors: A Combinatorial Approach Toward Esterase Mimics

Abstract

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.

Determination of Hemin‐Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach

“…The further steps typically required for staining as previously described for other systems could thus be omitted 77. 79, 80, 83 Control experiments with blank beads (no peptide bound) and unspecific bead‐bound peptides showed that the observed color was indeed due to selective binding of hemin by particular peptide sequences and not, for example, by the bead material itself. The peptides each also contained a C‐terminal amino acid linker of six residues (BBBBRM) providing good accessibility for hemin, thus supporting specific binding.…”

“…Positive beads were collected and analyzed by two methods: PED‐MALDI TOF mass spectrometry as introduced previously by Pei et al78 and direct on‐bead sequencing by automated Edman degradation 79. 80 The latter was used as a control to evaluate whether cysteine‐containing sequences can be found by the PED‐MS method, which had previously been applied only to non‐cysteine‐containing libraries. In the past five years, in fact, only a few combinatorial library approaches in which cysteines have been included at partially randomized positions in peptide sequences have been described 79.…”

“…In the past five years, in fact, only a few combinatorial library approaches in which cysteines have been included at partially randomized positions in peptide sequences have been described 79. 80, 84 Both sequencing methods gave the expected results, although the costs for automated Edman degradation hugely exceeded those for PED‐MS analyses, although the latter technique failed more often in complete identification of sequences, due to fragmentation or other effects during MS analysis.…”

“…78 In addition, Fmoc‐Cys(Trt) was incorporated at position P0 79. 80 The side chains of the trifunctional amino acids were protected as follows: Asn(Trt), Asp(O t Bu), Arg(Pbf), Cys(Trt), Gln(Trt), Glu(O t Bu), His(Trt), Lys(Boc), Ser( t Bu), Thr(O t Bu), Tyr( t Bu), and Trp(Boc). Prior to the screening, in a micro‐BioSpin column (0.8 mL Bio‐Rad), 20–45 mg of the library was washed extensively with CH 2 Cl 2 , followed by MeOH and double‐distilled water (ddH 2 O) and blocked for 1 h with HBST buffer [HEPES (30 m M ), pH 7.4, NaCl (150 m M ), Tween 20 (0.01 %)] containing gelatin (0.1 %).…”

Tuning the coordination properties of multi-histidine peptides by using a tripodal scaffold: solution chemical study and catechol oxidase mimicking

“…15 Tripodal peptidomimetics are also suitable for this purpose. Several tripodal peptides with different platforms have been designed as receptors of either organic molecules 16 or metal ions. [17][18][19][20][21][22][23][24][25][26][27][28][29] These ligands have several advantages in addition to the pre-organized structure and enhanced chelate effect.…”