p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and G termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Synthesis of gadolinium-labeled shell-crosslinked nanoparticles for magnetic resonance imaging applications

Shell-crosslinked knedel-like nanoparticles (SCKs; knedel is a Polish term for dumplings) were derivatized with gadolinium Shell chelates and studied as robust magnetic-resonance-imaging-active structures with hydrodynamic diameters of 40 +/- 3 nm. SCKs possessing an amphiphilic core-shell morphology were produced from the aqueous assembly of diblock copolymers of poly(acrylic acid) (PAA) and poly(methyl acrylate) (PMA), PAA(52)-b-PMA(128), and subsequent covalent crosslinking by amidation upon reaction with 2,2'-(ethylenedioxy)bis(ethylamine) throughout the shell layer. The properties of these materials, including non-toxicity towards mammalian cells, non-immunogenicity within mice, and capability for polyvalent targeting, make them ideal candidates for utilization within biological systems. The synthesis of SCKs derivatized with Gd-III and designed for potential use as a unique nanometer-scale contrast agent for MRI applications is described herein. Utilization of an amino-functionalized diethylenetriaminepentaacetic acid-Gd analogue allowed for direct covalent conjugation throughout the hydrophilic shell layer of the SCKs and served to increase the rotational correlation lifetime of the Gd. In addition, the highly hydrated nature of the shell layer in which the Gd was located allowed for rapid water exchange; thus, the resulting material demonstrated large ionic relaxivities (39 s(-1) mM(-1)) in an applied magnetic field of 0.47 T at 40 degrees C and, as a result of the large loading capacity of the material, also demonstrated high molecular relaxivities (20 000 s(-1) mM(-1)).

NMR of conotoxins: structural features and an analysis of chemical shifts of post-translationally modified amino acids

Conotoxins are small conformationally constrained peptides found in the venom of marine snails of the genus Conus. They are usually cysteine rich and frequently contain a high degree of post-translational modifications such as C-terminal amidation, hydroxylation, carboxylation, bromination, epimerisation and glycosylation. Here we review the role of NMR in determining the three-dimensional structures of conotoxins and also provide a compilation and analysis of H-1 and C-13 chemical shifts of post-translationally modified amino acids and compare them with data from common amino acids. This analysis provides a reference source for chemical shifts of post-translationally modified amino acids. Copyright (C) 2006 John Wiley & Sons, Ltd.