- Synthetic catalytic pores
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Catalytic activity of a synthetic multifunctional pore is studied in large unilamellar vesicles under conditions where substrate and synthetic catalytic pore (SCP) approach the membrane either from the same side (cis catalysis) or from opposite sides (trans catalysis). A synthetic supramolecular rigid-rod β-barrel with excellent ion channel characteristics is identified as SCP using 8-acetoxypyrene-1,3,6-trisulfonate (AcPTS) as model substrate. The key finding is that application of supportive membrane potentials increases the initial velocity of AcPTS esterolysis (v0). This results in an increase of Vmax beyond experimental error (+30%), whereas KM increases less significantly. Long-range electrostatic steering by the membrane potential, possibly guiding substrates into the transmembrane catalyst and, more importantly, accelerating product release (foff = 1.3) is discussed as one possible explanation of this global reduction of catalyst saturation. Control experiments show, inter alia, that similarly strong changes do not occur with opposing membrane potentials. Copyright
- Sakai, Naomi,Sorde, Nathalie,Matile, Stefan
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- Comparing dendritic with linear esterase peptides by screening SPOT arrays for catalysis
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Fluorescence screening of a 96-membered SPOT library of histidine containing dendritic and linear peptides revealed the remarkable esterolytic activity of short histidine oligomers that show catalytic proficiencies within one order of magnitude of histidine-containing esterase peptide dendrimers. The Royal Society of Chemistry 2010.
- Biswas, Rasomoy,Maillard, Noelie,Kofoed, Jacob,Reymond, Jean-Louis
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- Synthesis and activity of histidine-containing catalytic peptide dendrimers
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Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser (His = histidine, Ser = Serine, Dap = diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium (Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of the original third generation dendrimer A3 ((Ac-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser) 2Dap-His-Ser-NH2) were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against alanine (A3A), β-alanine (A3B), or threonine (A3C) improved catalytic efficiency. Substrate binding was correlated with the total number of histidines per dendrimer, with an average of three histidines per substrate binding site. The catalytic rate constant kcat depended on the placement of histidines within the dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant A3C ((Ac-His-Thr)8(Dap-His- Thr)4(Dap-His-Thr)2Dap-His-Thr-NH2), with kcat = 1.3 min-1, kcat/kuncat = 90′000, KM = 160 μM for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration of 18′000 per catalytic site and a 5-fold improvement over the original sequence A3.
- Delort, Estelle,Nguyen-Trung, Nhat-Quang,Darbre, Tamis,Reymond, Jean-Louis
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- Fluorescent ADP sensing in physiological conditions based on cooperative inhibition of a miniature esterase
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This study shows that a cationic esterase mimic, which is responsible for the catalytic hydrolysis of a negatively charged fluorogenic ester, displays different inhibition behaviors in the presence of similar anionic metabolites in water and at physiological pH. On the contrary of the other negatively charged inhibitors tested, ADP strongly slowed down the formation of the fluorescent product by virtue of cooperative inhibition, hence allowing its spectroscopic or visual detection. Our sensor or its derivatives may find future use in biological applications, such as protein kinase activity detection. Copyright
- Vial, Laurent,Dumy, Pascal
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- A combinatorial approach to catalytic peptide dendrimers
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Exploring the structural diversity of peptide dendrimers as synthetic protein models: A 65536-membered combinatorial peptide-dendrimer library was prepared by split-and-mix techniques on beads (see picture). The library was screened and revealed peptide dendrimers that catalyze fluorogenic ester hydrolysis and peptide dendrimers that bind to vitamin B12.
- Clouet, Anthony,Darbre, Tamis,Reymond, Jean-Louis
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- Design and high-resolution structure of a β3-peptide bundle catalyst
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Despite the widespread exploration of α-peptides as catalysts, there are few examples of β-peptides that alter the course of a chemical transformation. Our previous work demonstrated that a special class of β3-peptides spontaneously self-assembles in water into discrete protein-like bundles possessing unique quaternary structures and exceptional thermodynamic stability. Here we describe a series of β3-peptide bundles capable of both substrate binding and chemical catalysis ester hydrolysis. A combination of kinetic and high-resolution structural analysis suggests an active site triad composed of residues from at least two strands of the octameric bundle structure.
- Wang, Pam S. P.,Nguyen, Jennifer B.,Schepartz, Alanna
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- A peptide dendrimer enzyme model with a single catalytic site at the core
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Catalytic esterase peptide dendrimers with a core active site were discovered by functional screening of a 65 536-member combinatorial library of third-generation peptide dendrimers using fluorogenic 1-acyloxypyrene-3,6,8- trisulfonates as substrates. In the best catalyst, RMG3, ((AcTyrThr) 8(DapTrpGly)4-(DapArgSerGly)2DapHisSerNH 2), ester hydrolysis is catalyzed by a single catalytic histidine residue at the dendrimer core. A pair of arginine residues in the first-generation branch assists substrate binding. The catalytic proficiency of dendrimer RMG3 (kcat/KM = 860 M-1 min -1 at pH 6.9) per catalytic site is comparable to that of the multivalent esterase dendrimer A3 ((AcHisSer)8(DapHisSer) 4(DapHisSer)2DapHisSerNH2) which has fifteen histidines and five catalytic sites (Delort, E. et al. J. Am. Chem. Soc. 2004, 126, 15642-15643). Remarkably, catalysis in the single site dendrimer RMG3 is enhanced by the outer dendritic branches consisting of aromatic amino acids. These interactions take place in a relatively compact conformation similar to a molten globule protein as demonstrated by diffusion NMR. In another dendrimer, HG3 ((AcllePro)8(DaplleThr)4(DapHisAla) 2DapHisLeuNH2) by contrast, catalysis by a core of three histidine residues is unaffected by the outer dendritic layers. Dendrimer HG3 or its core HG1 exhibit comparable activity to the first-generation dendrimer A1 ((AcHisSer)2DapHisSerNH2). The compactness of dendrimer HG3 in solution is close to that a denatured peptide. These experiments document the first esterase peptide dendrimer enzyme models with a single catalytic site and suggest a possible relationship between packing and catalysis in these systems.
- Javor, Sacha,Delort, Estelle,Darbre, Tamis,Reymond, Jean-Louis
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- A strong positive dendritic effect in a peptide dendrimer-catalyzed ester hydrolysis reaction
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The contribution of the dendritic structure in catalysis of ester hydrolysis was investigated with a systematic peptide dendrimer series of increasing generation number (G1-G4) containing a catalytic consensus sequence His-Ser in all branches. A strong positive dendritic effect was observed with up to 100-fold increased histidine reactivity between G1 and G4. Kinetic studies and isothermal calorimetric titration experiments showed that the strong positive dendritic effect resulted from cooperativity between binding and catalysis. Copyright
- Delort, Estelle,Darbre, Tamis,Reymond, Jean-Louis
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- Triggered Release from Lipid Bilayer Vesicles by an Artificial Transmembrane Signal Transduction System
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The on-demand delivery of drug molecules from nanoscale carriers with spatiotemporal control is a key challenge in modern medicine. Here we show that lipid bilayer vesicles (liposomes) can be triggered to release an encapsulated molecular cargo in response to an external control signal by employing an artificial transmembrane signal transduction mechanism. A synthetic signal transducer embedded in the lipid bilayer membrane acts as a switchable catalyst, catalyzing the formation of surfactant molecules inside the vesicle in response to a change in external pH. The surfactant permeabilizes the lipid bilayer membrane to facilitate release of an encapsulated hydrophilic cargo. In the absence of the pH control signal, the catalyst is inactive, and the cargo remains encapsulated within the vesicle.
- Langton, Matthew J.,Scriven, Lorel M.,Williams, Nicholas H.,Hunter, Christopher A.
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p. 15768 - 15773
(2017/11/14)
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- Preparing method of 8-hydroxy-1,3,6-pyrene trisulphonic acid sodium salt
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The present invention discloses a preparing method of 8-hydroxy-1,3,6-pyrene trisulphonic acid sodium salt, pyrene is used as a raw material, sulfuric acid is used as a medium, after pyrene is dissolved, sulfur trioxide is added dropwise into the system, the system is stirred for reaction for 5 hours, the material is diluted in ice water to obtain paste-like pyrenetetrasulfonid acid tetrasodium salt, after filtering and draining, the pyrenetetrasulfonid acid tetrasodium salt is dissolved in water, added with a certain amount of sodium hydroxide, and kept warm at 100 DEG C for hydrolysis reaction to obtain the 8-hydroxy-1,3,6-pyrene trisulphonic acid sodium salt, the preparing method of the 8-hydroxy-1,3,6-pyrene trisulphonic acid sodium salt has the following benefits: after isolation and purification, the 8-hydroxy-1,3,6-pyrene trisulphonic acid sodium salt yield is 90% to 95%, the yield is high; the method is simple and efficient, the reaction steps are greatly reduced, cost is saved, and time is saved.
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Paragraph 0013; 0026; 0027
(2017/03/08)
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- Enzyme-artificial enzyme interactions as a means for discriminating among structurally similar isozymes
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We describe the design and function of an artificial enzyme-linked receptor (ELR) that can bind different members of the glutathione-S-transferase (GST) enzyme family. The artificial enzyme-enzyme interactions distinctly affect the catalytic activity of the natural enzymes, the biomimetic, or both, enabling the system to discriminate among structurally similar GST isozymes.
- Selvakumar, Karuthapandi,Motiei, Leila,Margulies, David
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supporting information
p. 4892 - 4895
(2015/05/05)
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- Combinatorial discovery of peptide dendrimer enzyme models hydrolyzing isobutyryl fluorescein
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Two 6750-membered one-bead-one-compound peptide dendrimer combinatorial libraries L (X4)8(LysX3)4(LysX 2)2LysX1 (X1-4 = 14 different amino acids or deletion, Lys = branching lysine residue) and AcL (with N-terminal acetylation) were prepared by split-and-mix solid phase peptide synthesis. Screening toward fluorogenic substrates for esterase and aldolase activities using the in silica off-bead assay (N. Maillard et al., J. Comb. Chem.2009, 11, 667-675) and bead decoding by amino acid analysis revealed histidine containing sequences active against fluorescein diacetate. Isobutyryl fluorescein, a related hydrophobic fluorogenic substrate, was preferentially hydrolyzed by dendrimers from library AcL containing hydrophobic residues such as AcH3 (AcHis)8(LysLeu)4(LysVal)2LysLysOH, compared to simple oligohistidine peptides as reference catalysts. Polycationic dendrimers from library L with multiple free N-termini such as H8 (His) 8(LysβAla)4(LysThr)2LysaProNH2 (aPro = (2S,4S)-4-aminoproline) showed stronger reactivity toward 8-acetoxypyrene-1,3,6-trisulfonate with partial acylation of N-termini. These experiments highlight the role of noncatalytic amino acids to determine substrate selectivity in peptide dendrimer esterase models.
- Maillard, Noelie,Biswas, Rasomoy,Darbre, Tamis,Reymond, Jean-Louis
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experimental part
p. 310 - 320
(2011/06/28)
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- Hydrolytic activity of histidine-containing octapeptides in water identified by quantitative screening of a combinatorial library
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A quantitative on-bead screening of a combinatorial octapeptide library revealed catalysts with hydrolytic activity in water. Histidine is an essential amino acid but the catalytic activity as well as the substrate binding affinity is also dependent on th
- Schmuck, Carsten,Michels, Ute,Dudaczek, Juergen
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body text
p. 4362 - 4368
(2009/12/25)
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- Esterolytic peptide dendrimers with a hydrophobic core and catalytic residues at the surface
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(3S)-4-(9-Fluorenylmethoxycarbonylamino)-3-methyl(allyloxycarbonyl) aminoethyloxyacetic acid (1) was prepared from (R)-3-aminopropane-1,2-diol and used as branching unit for the synthesis of second generation peptide dendrimers with six individually addressable variable amino acid positions. Three pairs of diastereomeric dendrimers were prepared bearing a common hydrophobic core and permutations of the catalytic triad amino acids aspartate, histidine and serine at the surface. Dendrimers with two surface histidine residues catalyzed the hydrolysis of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonates in aqueous buffer pH 6.0 with rate enhancement kcat/kuncat in the 10 3 range and Michaelis-Menten constants KM in the 10 -4 M range. Substrate recognition involves electrostatic interactions, as shown by competitive inhibition of catalysis observed with pyrene-1,3,6,8-tetrasulfonate. The 4-fold to 7-fold lowering in KM between the butyryl and nonanoyl esters in the most active dendrimers provides evidence for a hydrophobic component in substrate binding, which is absent in a closely related, less active diastereomeric peptide dendrimer.
- Clouet, Anthony,Darbre, Tamis,Reymond, Jean-Louis
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p. 1195 - 1204
(2007/10/03)
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- Selective catalysis with peptide dendrimers
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Peptide dendrimers incorporating 3,5-diaminobenzoic acid 1 as a branching unit (B) were prepared by solid-phase synthesis of ((Ac-A3) 2B-A2)2B-Cys-A1-NH2 followed by disulfide bridge formation. Twenty-one homo- and heterodimeric dendrimers were obtained by permutations of aspartate, histidine, and serine at positions A1, A2, and A3. Two dendrimers catalyzed the hydrolysis of 7-hydroxy-N-methyl-quinolinium esters (2-5), and two other dendrimers catalyzed the hydrolysis of 8-hydroxy-pyrene-1,3,6- trisulfonate esters (10-12). Enzyme-like kinetics was observed in aqueous buffer pH 6.0 with multiple turnover, substrate binding (KM = 0.1 -0.5 mM), rate acceleration (kcat/kuncat > 103), and chiral discrimination (E = 2.8 for 2-phenylpropionate ester 5). The role of individual amino acids in catalysis was investigated by amino acid exchanges, highlighting the key role of histidine as a catalytic residue, and the importance of electrostatic and hydrophobic interactions in modulating substrate binding. These experiments demonstrate for the first time selective catalysis in peptide dendrimers.
- Douat-Casassus, Celine,Darbre, Tamis,Reymond, Jean-Louis
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p. 7817 - 7826
(2007/10/03)
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- Cleft Formation upon Polymerization of Surfactant Vesicles
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Morphological consequences of the photopolymerization of vesicles prepared from (C18H37)2N+(CH3)CH2C6H4-p-CH-=CH2*Cl-, 1, have been investigated by using 8-hydroxy-1,3,6-pyrenetrisulfonate (POH) as a reporter group.Static and dynamic laser light scattering established the hydrodynamic radius and the molecular weight of 1 vesicles to be 425 +/-25 Angstroem and 2.3E7.Neither of these values changed upon polymerization.POH was shown to bind appreciably to 1 vesicles.Excitation of POH, following immediately its addition to nonpolymerized 1 vesicles, resulted in fluorescence emission with maxima at 440 and 520 nm.Incubation led to time-dependent changes of this spectra.Increasing incubation time of POH containing nonpolymerized 1 vesicles resulted in the gradual disappearance of the emission band at 520 nm and in the concomitant increase of the emission band at 440 nm.Fluorescence spectra of POH did not show any time-dependent changes following its addition to polymeryzed 1 vesicles.These results were interpreted in terms of the gradual penetration of POH into nonpolymerized 1 vesicles and in terms of long-term stabilization of POH in the clefts formed on the vesicle surface upon pulling the surfactant head groups together by photopolymerization.Differences between nonpolymerized and polymerized vesicles also manifested in the excited state protonation equilibria (POH)* (PO-)* + H+.A koff8 value of 4.3E9 s-1 was obtained in nonpolymerized vesicles immediately after the POH injection.Following a day of incubation no excited-state proton ejection could be observed in nonpolymerized 1 vesicles.Consequently, a koff* value of 6.1E9 s-1 was observed in nonpolymerized vesicles both immediately and 1 day subsequent to the addition of POH to polymerized 1 vesicles.Similar behavior has been observed for the steady-state (P) and nanosecond time-resolved polarizations, τR values, of POH, as well as that for the ground state PO- reprotonation (governed by Kon) in nonpolymerized and polymerized 1 vesicles.In nonpolymerized 1 vesicles, P values increased (from 0.12 to 0.19), τr was determined to be >/= 50 ns, and kon values (4E8 M-1 s-1) became unobservable after a day of incubation.In polymerized 1 vesicles, P values of 0.08, values τR of 4 ns, and kon values 8E8 M-1 s-1 remained unaffected by incubation.
- Nome, Faruk,Reed, Wayne,Politi, Mario,Tundo, Pietro,Fendler, Janos H.
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p. 8086 - 8093
(2007/10/02)
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