Marie A. Squire

Glycosylation of pramlintide: synthetic glycopeptides that display in vitro and in vivo activities as amylin receptor agonists.

Amylin is a 37-amino acid peptide that is co-secreted with insulin by pancreatic b-cells; it was first isolated from amyloid deposits in the pancreases of patients suffering from type II diabetes. Amylin acts as a partner hormone to insulin and is absent or significantly reduced in patients with type I diabetes, and furthermore, as with insulin, mealstimulated amylin secretion is markedly reduced in patients with type II diabetes. Administration of amylin either alone or in conjunction with insulin would therefore be expected to have beneficial effects for sufferers of either type I or II diabetes. However, amylin has particularly unfavourable physiochemical properties, such as poor solubility and a tendency to self-aggregate, which precludes its clinical use. A synthetic version of amylin with superior properties called pramlintide was, therefore, developed by Amylin Pharmaceuticals, in which comparison with the structure of rat amylin led to replacement of three amino acids present in human amylin (A25 and S28 and S29) with proline, and resulted in a peptide with improved physiochemical properties. Pramlintide (Symlin) was approved in 2005 by the FDA for use in US in patients with type I and II diabetes in conjunction with administration of prandial insulin to improve postprandial glycemic control. Pramlintide still has significant limitations, typical of other peptide drugs, including low circulatory half-life and poor solubility/bioavailability issues, which means that it currently has to be administered as a separate injection to insulin. For several years it has been proposed that glycosylation may have beneficial effects on the pharmacokinetic properties of peptide and protein drugs, for example, by improving solubility/increasing oral bioavailability and/or by increasing circulatory lifetime. Glycosylation of several synthetic peptides relevant to the treatment of diabetes has been attempted to improve their pharmacokinetic properties, including insulin, GLP-1, and exendin-4. Glycosylation of amylin or amylin analogues, such as pramlintide may, therefore, be expected to produce glycopeptides with superior properties. Native amylin itself circulates in nonglycosylated (~50 %) and also glycosylated forms in which O-glycans are attached to threonine residues near the N terminus. The latter are known to be biologically inactive, and it could, therefore, be assumed that glycosylation at either T6 or T9 completely ablates agonist activity. It is perhaps for this reason that there has been no reported synthesis or biological study of glycosylated amylin analogues to date. Nevertheless amylin comprises of multiple amino acids to which glycans might be attached. The potential benefit from site-specific glycosylation warranted an exploration of a structure–activity relationship. A program was initiated to study the effect of Nlinked glycosylation on the properties of pramlintide. Standard synthetic approaches to glycopeptides utilize either elaborate glycoamino acid building blocks during peptide synthesis or sequential modification of a glycopeptide chain with glycosyl transferases (GTases) to provide short nonnatural glycans. However, recent developments into the use of endo-b-N-acetylglucosaminidases (ENGases) have revealed their application to be a powerful method for the convergent assembly of complex glycoconjugates. Key recent developments in the field include the production of glycosynthase mutants of these enzymes, and access to N-glycan oxazolines as efficient donors, compounds that may now be accessed directly from the corresponding reducing sugars in water. Although it has been demonstrated that ENGases will in certain cases catalyse the transfer of N-glycan oligosaccharides to other substrates, in general a pre-existing GlcNAc residue is required at the site of the attachment of N-glycans. We elected to prepare two pramlintide glycopeptides in which a GlcNAc residue was attached to either the Asn residue at position 3 or 21 by solidphase peptide synthesis (SPPS, Scheme 1). Removal of the protecting groups from the sugar and amino acid side chains, cleavage from the resin, and cyclic disulfide formation produced two glycopeptides 3 and 4, with a GlcNAc at N3 and N21, respectively. These were [a] Dr. Y. Tomabechi, Dr. M. A. Squire, Prof. A. J. Fairbanks Department of Chemistry, University of Canterbury Private Bag 4800, Christchurch 8140 (New Zealand) E-mail : antony.fairbanks@canterbury.ac.nz [b] Dr. G. Krippner Baker IDI Heart and Diabetes Institute, 75 Commercial Road Melbourne, Victoria 3004 (Australia) [c] Dr. P. M. Rendle Callaghan Innovation, PO Box 31310, 69 Gracefield Road Lower Hutt 5040 (New Zealand) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303303.

Skin peptides of different life stages of Ewing's tree frog.

In frogs, an important mechanism of skin innate immunity against invading microbial pathogens is secretion of antimicrobial peptides from the specialized granular glands. Since these glands develop fully in skin dermis after completion of metamorphosis, they are small and immature in skin of larvae (tadpoles). Skin secretions vary among different life stages. Antimicrobial activity and peptide composition of natural mixture of skin peptides of three different life stages of New Zealand Ewings Tree Frog (Litoria ewingii), tadpoles, metamorphs and adults were analyzed. The peptide mixtures were collected from skin secretions and analyzed for activity against the standard reference bacterium, Escherichia coli (ATCC 25922). Their peptide components were analyzed using liquid chromatography mass spectrometry (LC-MS). The peptide mixture from adults and metamorphs contained the species-specific antimicrobial peptide uperin 7.1 and inhibited the growth of E. coli (ATCC 25922). In contrast, the peptide mixture of tadpoles did not inhibit the growth of E. coli (ATCC 25922). This peptide mixture did not contain uperin 7.1 but had peptides whose molecular masses did not correspond to molecular masses of any known frog antimicrobial peptides.

Breath testing and personal exposure—SIFT-MS detection of breath acetonitrile for exposure monitoring

Breath testing has potential for the rapid assessment of the source and impact of exposure to air pollutants. During the development of a breath test for acetonitrile using selected ion flow tube mass spectrometry (SIFT-MS) raised acetonitrile concentrations in the breath of volunteers were observed that could not be explained by known sources of exposure. Workplace/laboratory exposure to acetonitrile was proposed since this was common to the volunteers with increased breath concentrations. SIFT-MS measurements of acetonitrile in breath and air were used to confirm that an academic chemistry laboratory was the source of exposure to acetonitrile, and quantify the changes that occurred to exhaled acetonitrile after exposure. High concentrations of acetonitrile were detected in the air of the chemistry laboratory. However, concentrations in the offices were not significantly different across the campus. There was a significant difference in the exhaled acetonitrile concentrations of people who worked in the chemistry laboratories (exposed) and those who did not (non-exposed). SIFT-MS testing of air and breath made it possible to determine that occupational exposure to acetonitrile in the chemistry laboratory was the cause of increased exhaled acetonitrile. Additionally, the sensitivity was adequate to measure the changes to exhaled amounts and found that breath concentrations increased quickly with short exposure and remained increased even after periods of non-exposure. There is potential to add acetonitrile to a suite of VOCs to investigate source and impact of poor air quality.