Paul W. R. Harris

A one-pot approach to neoglycopeptides using orthogonal native chemical ligation and click chemistry.

The powerful combination of native chemical ligation and click chemistry has been used to affect a one-pot synthesis of neoglycopeptides from propargyl-containing peptides using GalNAc-N(3) as the glycan component. A versatile chemical toolkit for the fully convergent synthesis of neoglycoproteins using click chemistry, native chemical ligation, and kinetically controlled ligation is thus demonstrated.

Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Ruthenium-catalysed ortho vinylation of aromatic ketones with alkynes; unexpected cyclopentaannulation

Abstract Ruthenium-catalysed coupling of aromatic ketones with alkynylsilanes yielded in most cases ortho vinylation adducts in high yield. The predominant stereochemistry of the newly introduced double bond was E in all but a few cases. In contrast, 1-acetylnaphthalene underwent a one-pot insertion–cyclisation sequence yielding cyclopenta[ a ]naphthalene derivatives. A bis acetylene gave both mono and bis insertion products.

Ruthenium-catalysed ortho alkylation of hydroxyacetophenones; the functionalisation of ring C aromatic diterpenoids

Abstract Ortho C-H bond coupling of some 2-alkoxyacetophenones with olefins catalysed by ruthenium complexes results in a high yield of the ortho alkylated product, providing that a suitable protecting group is employed. No such protection was required for a para-alkoxy group; an activating effect was also observed. Bicyclic and tricyclic analogues react similarly.

Direct peptide lipidation through thiol-ene coupling enables rapid synthesis and evaluation of self-adjuvanting vaccine candidates.

A radical lipidation: Application of a novel thiol-ene lipidation enables the one-step synthesis of self-adjuvanting antigenic peptides as vaccine candidates. The resultant monoacyl lipopeptides are shown to activate monocytes in a robust manner.

Peripheral administration of a novel diketopiperazine, NNZ 2591, prevents brain injury and improves somatosensory-motor function following hypoxia–ischemia in adult rats

The current study describes the neuroprotective effects of an endogenous diketopiperazine, cyclo-glycyl-proline (cyclic GP), in rats with hypoxic-ischemic brain injury and the pre-clinical development of an analogue, cyclo-L-glycyl-L-2-allylproline (NNZ 2591), modified for improved bioavailability. The compounds were given either intracerebroventricularly or subcutaneously 2h after hypoxia-ischemia. Histology, immunohistochemistry and behavior were used to evaluate treatment effects. The central uptake of NNZ 2591 was also examined in normal and hypoxic-ischemic injured rats by HPLC-mass spectrometry. Central administration of cyclic GP or NNZ 2591 reduced the extent of brain damage in the lateral cortex, the hippocampus and the striatum (p<0.001), with NNZ 2591 being more potent. NNZ 2591 was stable in the plasma and crossed the blood-brain barrier independent of hypoxic-ischemic injury. The level of NNZ 2591 in the CSF was maintained for 2 h after a single subcutaneous dose, and modest neuroprotection was seen after a bolus subcutaneous administration (overall p<0.001). Treatment with NNZ 2591 for 5 d subcutaneously improved somatosensory-motor function (p<0.05) and long-term histological outcome (overall p<0.0001). NNZ 2591 treatment not only reduced both caspase-3 mediated apoptosis and microglial activation but also enhanced astrocytic reactivity, which may mediate its protective effect. The pharmacokinetic profile and potent long-term protective effects of NNZ 2591 suggests its utility for the treatment of ischemic brain injury and other neurological conditions requiring chronic intervention.

Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies

Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.

The Synthesis of Phosphopeptides Using Microwave-assisted Solid Phase Peptide Synthesis

A series of phosphorylated peptides were synthesised using microwave mediated solid phase peptide synthesis. Acidic cleavage of peptides from the solid support using microwave irradiation often resulted in reattachment of the phosphate benzyl protecting group to the peptide chain. However for most phosphopeptide sequences performing the cleavage reaction at room temperature in order to minimize this undesired alkylation was successful. Notably for phosphopeptides containing a methionine residue flanking the phosphorylated residue (for serine and threonine) the trifluoroacetic acid mediated cleavage afforded the benzylated side product as a major component. This detrimental process was not observed for a corresponding tyrosine containing sequence.

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.

Synthesis of MUC1 Neoglycopeptides using efficient microwave-enhanced chaotrope-assisted click chemistry

The first synthesis of click neoglycopeptide analogues of the biologically relevant MUC1 sequence is reported. In the process, microwave-enhanced chaotrope-assisted click reaction conditions that may be used on a routine basis for the synthesis of click peptide conjugates have been developed. The convergent route for the synthesis of neoglycopeptides using these reaction conditions enables the facile, rapid, and highly efficient preparation of focused neoglycopeptide libraries of defined chemical structure for biological evaluation.