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Biomedical subjects

Jean-Louis Reymond

Publications and source records attributed to Jean-Louis Reymond.

At least 19 recordsLinked to original sources
PubMed2007

Fluorogenic substrates for lipases, esterases, and acylases using a TIM-mechanism for signal release.

3-Acyloxyl-2-oxopropyl ethers of umbelliferone were investigated as new fluorogenic substrates for lipases and esterases. The aliphatic primary alcohol-leaving group released the fluorescent product umbelliferone by an enolization/beta-elimination reaction similar to the triose phosphate isomerase (TIM) reaction. A similarly designed phenylacetamide provided a fluorescent probe for penicillin G acylase, whereby the enolization/beta-elimination sequence from the intermediate aminoketone was very fast and spontaneous even under acidic conditions. The corresponding epoxyketone was not fluorogenic with epoxide hydrolases (EH). These substrates represent periodate-free Clips-otrade mark substrates.

Acetamides↗
PubMed2006

A red-fluorescent substrate microarray for lipase fingerprinting.

A lipase substrate microarray was obtained by printing aliphatic C2-C12 monoesters of (5R)- and (5S)-3-(5,6-dihydroxyhexyloxy)benzaldehyde by reductive alkylation on amine-functionalized glass slides coated with bovine serum albumin and a short PEG linker. The microarray features 12 substrates and their 66 possible binary mixtures spotted in a 9 x 36 spot array. Lipase reactions are detected by chemoselective NaIO(4)-oxidation of the 1,2-diol hydrolysis product to form an aldehyde, which is then tagged with the red-fluorescent dye rhodamine B sulfohydrazide . Specific fingerprints are produced by active enzymes. These experiments provide the first example of lipase fingerprinting using microarrays.

Cluster Analysis↗
PubMed2006

Artificial aldolases from peptide dendrimer combinatorial libraries.

Peptide dendrimers were investigated as synthetic models for aldolase enzymes. Combinatorial libraries were prepared with aldolase active residues such as lysine and proline placed at the dendrimer core or near the surface. On-bead selection for aldolase activity was carried out using the dye-labelled 1,3-diketone 1a, suitable for covalent trapping of enamine-reactive side-chains, and the fluorogenic enolization probe 6. Aldolase dendrimers catalyzed the aldol reaction of acetone, dihydroxyacetone and cyclohexanone with nitrobenzaldehyde. Much like enzymes, the dendrimers exhibited strong aldolase activity in aqueous medium, but were also active in organic solvent. Dendrimer-catalyzed aldol reactions reached complete conversion in 3 h at 25 degrees C with 1 mol% catalyst and gave aldol products with up to 65% ee. A positive dendritic effect in catalysis was observed with both lysine and proline based aldolase dendrimer catalysts.

Amino Acids↗
PubMed2006

Synthesis and activity of histidine-containing catalytic peptide dendrimers.

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), beta-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/k(uncat) = 90'000, KM = 160 microM 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.

Binding Sites↗
PubMed2006

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

Dinitrobenzenes↗
PubMed2006

Dual mechanism of zinc-proline catalyzed aldol reactions in water.

The aldol reaction of acetone with aldehydes in aqueous medium under catalysis by zinc-proline (Zn(L-Pro)2) and secondary amines such as proline, (2S,4R)-4-hydroxyproline (Hyp) and (S)-(+)-1-(2-pyrrolidinomethyl)pyrrolidine (PMP) is shown to proceed by an enamine mechanism, as evidenced by reductive trapping of the iminium intermediate, while the aldol reaction of dihydroxyacetone (DHA) under catalysis by zinc-proline and by general bases such as N-methylmorpholine (NMM) is shown to occur under rate-limiting deprotonation of the alpha-carbon and formation of an enolate intermediate.

Journal Article↗
PubMed2006

Combinatorial synthesis, selection, and properties of esterase peptide dendrimers.

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.

Arylsulfonates↗
PubMed2006

Peptide dendrimers as artificial enzymes, receptors, and drug-delivery agents.

The dendritic architecture applied to peptides provides a practical entry into globular macromolecules resembling proteins. A modular design was chosen using a divergent synthesis on solid support alternating proteinogenic alpha-amino acids with branching diamino acids, producing peptide dendrimers with a molecular weight of 3-5 kDa. Initial studies focused on models for hydrolases and produced esterase peptide dendrimers featuring histidine as the key catalytic residue. Variations of amino acid composition and the branching diamino acid led to enantioselective catalysts. Rate accelerations of k(cat)/k(uncat) = 90,000 were obtained when the design was changed to monomeric peptide dendrimers alternating two amino acids with the branching unit. A combinatorial approach was developed allowing for the preparation of large libraries (>60,000 members), which were screened for B12 binding and catalytic activity. The peptide dendrimers were also investigated for drug delivery. Glycopeptide dendrimers conjugated to colchicine selectively inhibited the proliferation of targeted cells, whereas colchicine alone displayed high toxicity.

Combinatorial Chemistry Techniques↗
PubMed2006

Inflammatory destruction of elastic fibers in acquired cutis laxa is associated with missense alleles in the elastin and fibulin-5 genes.

Cutis laxa (CL) is a condition characterized by redundant, pendulous, and inelastic skin. Acquired CL has been reported in patients with inflammatory diseases. The goal of this study was to investigate whether genetic lesions predispose patients to the development of acquired CL. We report a patient who developed CL following a Toxocara canis parasitism. He later had an aortic root aneurysm that required surgical correction. Histological evaluation showed inflammation followed by destruction of elastic fibers in both the skin and the aorta. Mutational analysis showed that the patient was heterozygous for an inherited fibulin-5 (FBLN5) allele G202R and compound heterozygous for elastin (ELN) alleles A55V and G773D. Western blotting indicated abnormal proteolytic processing of tropoelastin (TE) in patient fibroblasts. The FBLN5 202R allele on the other hand led to increased interaction of FBLN5 and TE and increased deposition of insoluble ELN partially rescuing the deficiency conferred by ELN mutation G773D. We demonstrated that the interaction of ELN and FBLN5 alleles results in elastic fibers susceptible to inflammatory destruction. These results suggest that the pathogenesis of acquired CL involves an underlying genetic susceptibility and highlight the importance of molecular genetic analysis in patients with idiopathic connective tissue disorders.

Adult↗
PubMed2005

Dendrimers as artificial enzymes.

Dendrimers are regular tree-like macromolecules accessible by chemical synthesis from a variety of building blocks. Their topology enforces a globular shape that offers a unique opportunity to design artificial enzymes. Catalytic groups such as metal complexes and cofactors can be placed at the dendrimer core to exploit microenvironment and selectivity effects of the dendritic shell. In a second approach, attaching catalytic groups in multiple copies at the end of the dendritic branches may lead to cooperativity effects. Finally, exploration of dendritic structural space by screening combinatorial libraries of peptide dendrimers for catalytic activity can lead to discovery of functional dendrimers with enzyme-like properties, in a process mimicking natural selection.

Catalysis↗
PubMed2005

Inhibition of mitosis by glycopeptide dendrimer conjugates of colchicine.

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (beta-glucose, alpha-galactose, alpha-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC50 > 1 microM) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.

Animals↗
PubMed2005

Protease profiling using a fluorescent domino peptide cocktail.

Five hexapeptides were prepared containing, in a domino-type arrangement, all 25 possible dipeptides between (1) aromatic, (2) hydrophobic, (3) positively charged, (4) negatively charged, and (5) small and polar amino acids. The peptides were fluorescence labeled at the N-terminus with a (7-coumaryl)oxyacetyl group, allowing the selective detection of N-terminal cleavage products. The five peptides were used as a cocktail reagent in an HPLC analysis. The cocktail produced specific cleavage patterns, or fingerprints, for a variety of proteases. This domino peptide cocktail can be used as a general reagent for protease identification and functional profiling.

Chromatography, High Pressure Liquid↗
PubMed2005

Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling.

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.

Antibodies, Catalytic↗
PubMed2005

A high-throughput, low-volume enzyme assay on solid support.

A high-throughput enzyme assay is described that uses 1 microL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.

Autoanalysis↗
PubMed2004

A strong positive dendritic effect in a peptide dendrimer-catalyzed ester hydrolysis reaction.

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.

Calorimetry↗