Competitive pathways for peptide deamidation

Asparagine (Asn) residues spontaneously – yet non-enzymatically – deamidate to form aspartate under physiological conditions, causing time-dependent changes in the conformation of proteins, limiting their lifetime [1]. The 'molecular clocks' hypothesis [2], suggests that deamidation is a biological molecular timing mechanism that could be set to any desired time interval by genetic control of the protein structure and the immediate environment of the Asn residue. The fact that deamidation occurs over a wide range of biologically relevant time intervals suggests that different mechanisms may be at play. To date deamidation is believed to occur over a succinimide-mediated pathway [3]. A novel route leading to the succinimide intermediate via tautomerization of the Asn side chain amide functionality was recently proposed [4,5]. The current study introduces a new 'competing' route for the deamidation of asparagine residues. The aim is to comparatively analyze the feasibility of this new mechanism against the traditional succinimide route, taking into account the catalytic effect of the solvent environment. For this purpose, QM dynamics and meta-dynamics calculations were performed on a model peptide placed in a periodic water box. These results will identify the lowest energy pathway for asparagine deamidation and will serve as a stepping stone for QM/MM calculations of Asn deamidation in proteins

Stability enhancing N-Terminal PEGylation of oxytocin exploiting different polymer architectures and conjugation approaches

Oxytocin, a cyclic nine amino acid neurohypophyseal hormone therapeutic, is effectively used in the control of postpartum hemorrhaging (PPH) and is on the WHO List of Essential Medicines. However, oxytocin has limited shelf life stability in aqueous solutions, particularly at temperatures in excess of 25 °C and injectable aqueous oxytocin formulations require refrigeration (<8 °C). This is particularly problematic in the hot climates often found in many developing countries where daytime temperatures can exceed 40 °C and where reliable cold-chain storage is not always achievable. The purpose of this study was to develop N-terminal amine targeted PEGylation strategies utilizing both linear PEG and polyPEG “comb” polymers as an effective method for stabilizing solution formulations of this peptide for prolonged storage in the absence of efficient cold-chain storage. The conjugation chemistries investigated herein include irreversible amine targeted conjugation methods utilizing NHS ester and aldehyde reductive amination chemistry. Additionally, one reversible conjugation method using a Schiff base approach was explored to allow for the release of the native peptide, thus, ensuring that biological activity remains unaffected. The reversibility of this approach was investigated for the different polymer architectures, alongside a nonpolymer oxytocin analogue to monitor how pH can tune native peptide release. Elevated temperature degradation studies of the polymer conjugates were evaluated to assess the stability of the PEGylated analogues in comparison to the native peptide in aqueous formulations to mimic storage conditions in developing nations and regions where storage under appropriate conditions is challenging

A spoonful of sugar: the application of glycopolymers in therapeutics

Glycopolymers, synthetic polymers displaying carbohydrate moieties, have been linked to many potential applications at the biology–chemistry interface. One area that holds particular promise is the employment of glycopolymers as vehicles for therapeutics or as therapeutics themselves. This review summarises some of the more prominent examples as well as those in the early stages of development

Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids

This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols

Investigation of the reaction of α-Thioamides, α-esters and α–nitriles with N-halosuccinimides

Investigation of the reaction of α-thioamides, α-esters and α-nitriles with NBS and NCS is described. The scope of this stereoselective oxidative transformation to the β- haloacrylamides, β-acrylates and β–acrylonitriles has been determined. A mechanistic rationale to explain the observed differences in reactivity between the amide, ester and nitrile series is proposed

The structure of latherin, a surfactant allergen protein from horse sweat and saliva

Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting microbial biofilms. How these proteins work as surfactants while remaining soluble and cell membrane-compatible is not known. Nor have their structures previously been reported. We have used protein nuclear magnetic resonance spectroscopy to determine the conformation and dynamics of latherin in aqueous solution. The protein is a monomer in solution with a slightly curved cylindrical structure exhibiting a ‘super-roll’ motif comprising a four-stranded anti-parallel β-sheet and two opposing α-helices which twist along the long axis of the cylinder. One end of the molecule has prominent, flexible loops that contain a number of apolar amino acid side chains. This, together with previous biophysical observations, leads us to a plausible mechanism for surfactant activity in which the molecule is first localized to the non-polar interface via these loops, and then unfolds and flattens to expose its hydrophobic interior to the air or non-polar surface. Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported. Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far

Polymeric organoiron compounds with carcinostatic properties (branched hydrazone linkers)

The insufficient efficaciousness of most currently used anticancer drugs has prompted worldwide efforts to reduce toxic and resistance effects, improve overall bioavailability, and widen the therapeutic window. A particularly promising technology to this end rests on the concept of polymer-drug conjugation, in which the bioactive agent is bound to a meticulously designed macromolecular water-soluble carrier through a biofissionable link. Drug release in the cancerous cell, strongly pH dependant, proceeds hydrolytically in the acidic intracellular compartment, and this represents an advanced drug delivery method in cancer chemotherapy. The synthesis of water-soluble macromolecular anticancer drugs composed of a polymeric carrier to which the antineoplastic agents are tied via biodegradable hydrazone links were investigated in this project. Carriers were synthesized essentially by polyaddition and ring-opening methods, and polycondensation process was utilized, refined and routinely used. Polyaspartamides derived from polysuccinimide by aminolytic ring-opening was the parent carrier’s structure, allowing for: a) A non-immunogenic and non-toxic chain construction, which was amenable to biodegradation and ensured catabolic elimination of the duly fragment polymer upon drug release; b) A highly flexible backbone and the presence of intrachain-type or side group-attached solubilizing groups, which ensured conjugate solution in aqueous media required for rapid dissipation in the central circulation system, even if the conjugated drug itself does not possess water solubility; and; c) The presence of functional groups as binding sites, represented by the hydrazone entity, which ensured drug attachment and release, was introduced by treatment of polysuccinimide with hydrazine hydrate under specially developed experimental conditions, followed by treatment with selected, functionally active amines providing the aforementioned structural features. Drug systems were modified so as to contain carbonyl functionality, the crucial reaction site in this hydrazone linking process, and bioactive aldehydes, such as ferrocenylpropenal. A cinnamaldehyde was the primary drug model. In order to illustrate the multidrug-binding capacity of the polyaspartamide type carriers, and at the same time ensuring target-specific drug delivery, folic acid, a potential cell entry facilitator, was co-conjugated to selected polymeric conjugate containing ferrocenylpropenal. Cell carrier and conjugate polymers were purified, fractionated by aqueous phase dialysis in membrane tubing with 12 000 – 14 000 molecular – mass cut - off, and isolated by freeze-drying in ultimate yields of 45 – 80 % as water-soluble materials; and they were structurally characterized by spectroscopic techniques. Inherent viscosities were in the range of 8 – 36 mL g-1. The resulting cinnamaldehyde, curcumin and iron contents of the conjugates were in the range of 4 - 7 %, 10 – 14 % and 1.5 – 2.8 % respectively. In vitro experiments done under buffered solution (performed in polymer laboratory of school of chemistry of the university of the Witwatersrand) showed the released of drugs in cancer cell's pH (pH<7). The results of these tests suggest that in acidic environment PSI-hydrazine carriers drugs systems can release active drug such as ferrocenylpropenal, and on the other hand the polymers drugs systems showed higher stabilities under neutral conditions. Therefore drugs released under pH control can play an important role in future cancer therapy

Two-Dimensional Controlled Syntheses of Polypeptide Molecular Brushes via N-Carboxyanhydride Ring-Opening Polymerization and Ring-Opening Metathesis Polymerization.

Well-defined molecular brushes bearing polypeptides as side chains were prepared by a "grafting through" synthetic strategy with two-dimensional control over the brush molecular architectures. By integrating N-carboxyanhydride ring-opening polymerizations (NCA ROPs) and ring-opening metathesis polymerizations (ROMPs), desirable segment lengths of polypeptide side chains and polynorbornene brush backbones were independently constructed in controlled manners. The N2 flow accelerated NCA ROP was utilized to prepare polypeptide macromonomers with different lengths initiated from a norbornene-based primary amine, and those macromonomers were then polymerized via ROMP. It was found that a mixture of dichloromethane and an ionic liquid were required as the solvent system to allow for construction of molecular brush polymers having densely-grafted peptide chains emanating from a polynorbornene backbone, poly(norbornene-graft-poly(β-benzyl-l-aspartate)) (P(NB-g-PBLA)). Highly efficient postpolymerization modification was achieved by aminolysis of PBLA side chains for facile installment of functional moieties onto the molecular brushes

Catalytic electrophilic halogenation of silyl-protected and terminal alkynes: trapping gold(I) acetylides vs. a bronsted acid-promoted reaction

In the presence of a cationic gold(I) catalyst and N-halosuccinimide, both trimethylsilyl-protected and terminal alkynes are converted into alkynyl halides. Further experiments showed that silyl-protected alkynes undergo electrophilic iodination and bromination under Brønsted acid catalysis, whilst terminal alkynes require a cationic gold catalyst. The former reactions probably proceed via activation of the electrophile, whilst the latter reactions proceed via a gold(I) acetylide intermediate. Gold-catalysed halogenation was further combined with gold-catalysed hydration and subsequent annulation to provide convenient routes to iodomethyl ketones and five-membered aromatic heterocycles