Morten Meldal

Pega: a flow stable polyethylene glycol dimethyl acrylamide copolymer for solid phase synthesis.

Abstract A copolymer of bisacrylamido polyethylene glycol, monoacrylamido polyethylene glycol and N,N-dimethyl acrylamide was synthesized by radical polymerization and characterized for peptide synthesis by preparation of the difficult test sequence 65–74 from the acyl carrier protein. Reaction times were recorded and compared with synthesis on kieselguhr supported polydimethyl acrylamide gel.

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases : multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.

POEPOP and POEPS: Inert polyethylene glycol crosslinked polymeric supports for solid synthesis

Two types of macromonomers were synthesized by reacting mono/di sodium derivatives of polyethylene glycol1500 with vinylbenzyl chloride and epichlorohydrin. These monomers were bulk homo-polymerized by freeradical or anionic polymerization, respectively, to afford two different types of crosslinked polymeric supports in high yield. The resins were tested for solid-phase peptide synthesis by standard Fmoc/OPfp/Dhbt method, using a Rink amide linker. The peptides were cleaved from the support using 95% TFA-H2O in high yield and purity. The pure peptides were characterized by MALDI-TOF mass spectrometry and amino acid analysis.

Epitope affinity for MHC class I determines helper requirement for CTL priming

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC–restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.

Azido Acids in a Novel Method of Solid-Phase Peptide Synthesis.

Azido acids were produced from α-branched acids by α-bromination with NBS followed by substitution with sodium azide and the products were used in a novel method of solid-phase synthesis. The azido acids were transformed into the highly activated acid chlorides and used synthesis of extremely hindered peptides containing up to four successive diphenyl glycine or Aib residues. By reaction of the genetically encoded amino acids with TfN3 and then SOCl2 they were transformed into α-azido acid chlorides used in solid-phase peptide synthesis without racemization.

Glycopeptide and Oligosaccharide Libraries.

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.

Peptidotriazoles: Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions on Solid-Phase

[1,2,3]-Triazoles are important pharmacophores in medicinal chemistry and it is therefore important to develop synthetic methods for triazoles that are both mild, high yielding and applicable to combinatorial synthesis. [1,2,3]-Triazoles are typically prepared by refluxing an alkyne and an azide in toluene (110 °C), but the 1,3-dipolar cycloaddition has also been performed at lower temperatures by using sodium acetylide [1], lithium and magnesium acetylide [2,3] with varying success. The present work describes the preparation of [1,2,3]-triazoles at 25 °C on solid-phase with high yields (80–95%), and is compatible with Fmoc chemistry.

Pentafluorophenyl esters for temporary carboxyl group protection in solid phase synthesis of N-linked glycopeptides.

The compound Ac3GlcNAcβ1-NH-Fmoc (3) was synthesized and transformed into the β-glucosyl amine (1) which was subsequently acylated with Fmoc-Asp(Cl)-O-Pfp (5) prepared from the readily available Fmoc-Asp(O-tBu)-O-Pfp (4). The resulting Fmoc-Asn(Ac3GlcNAcβ1-N-)-O-Pfp (6) was used as a building block in the solid phase synthesis of an 11 residue glycopeptide fragment of the enzyme glucoamylase (AMG).

A PEGA resin for use in the solid-phase chemical–enzymatic synthesis of glycopeptides

The successful application of a new resin consisting of beaded polyethylene glycol polyacrylamide copolymer 5(PEGA1900) as a solid support for the chemical–enzymatic synthesis of glycopeptides is reported; the resin is mechanically stable, yet highly swelling in both organic solvents and aqueous buffers.