Design strategies for controlling the molecular weight and rate using reversible addition-fragmentation chain transfer mediated living radical polymerization

Living radical polymerization has allowed complex polymer architectures to be synthesized in bulk, solution, and water. The most versatile of these techniques is reversible addition-fragmentation chain transfer (RAFT), which allows a wide range of functional and nonfunctional polymers to be made with predictable molecular weight distributions (MWDs), ranging from very narrow to quite broad. The great complexity of the RAFT mechanism and how the kinetic parameters affect the rate of polymerization and MWD are not obvious. Therefore, the aim of this article is to provide useful insights into the important kinetic parameters that control the rate of polymerization and the evolution of the MWD with conversion. We discuss how a change in the chain-transfer constant can affect the evolution of the MWD. It is shown how we can, in principle, use only one RAFT agent to obtain a poly-mer with any MWD. Retardation and inhibition are discussed in terms of (1) the leaving R group reactivity and (2) the intermediate radical termination model versus the slow fragmentation model. (c) 2005 Wiley Periodicals, Inc.

Single turn peptide alpha helices with exceptional stability in water

Cyclic pentapepticles are not known to exist in a-helical conformations. CD and NMR spectra show that specific 20-membered cyclic pentapepticles, Ac-(cyclo-1,5) [KxxxD]-NH2 and Ac-(cyclo-2,6)R[KxxxD]-NH2, are highly a-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest a-helical peptides in water.

Topological side-chain classification of beta-turns: Ideal motifs for peptidomimetic development

beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi 2, psi 2, phi 3 and psi 3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C-alpha-C-beta vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C-alpha-C-beta vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Epidermal penetration of a therapeutic peptide by lipid conjugation; Stereo-selective peptide availability of a topical diastereomeric lipopeptide

In inflammatory disorders (e.g. psoriasis), local concentrations of human neutrophil elastase (HNE), also known as polymorphonuclear leukocyte elastase (HLE), possibly overwhelm its natural inhibitors leading to extracellular matrix degradation. Elevated levels of HNE have been reported in a variety of inflammatory disorders, including psoriasis. Peptidic HNE inhibitors have a common hydrophobic sequence (Ala-Ala-Pro-Val). This peptide sequence inhibits HNE competitively; however the stratum corneum presents an effective barrier to the delivery of this tetrapeptide across the skin. The current work investigates the delivery of the modified peptide whereby the tetrapeptide was lipidated to enhance its ability to penetrate the stratum The tetrapeptide Was Coupled to a racaemic mixture of a short chain lipoamino acid (LAA) resulting in two diastereomers of the lipoamino acid-modified tetrapeptide. The penetration of the lipopeptide mixture was assessed across human epidermis in vitro. The percentage of applied dose penetrating to the receptor over 8 h following administration was 2.53% for the D-LAA conjugate and 1.47% for the L-diastereomer, compared to 0% for the peptide. The D-diastereomer appears to be relatively stable but the L-diastereomer appears to degrade releasing possibly the tetrapeptide and peptide fragment(s). Therefore the results clearly indicate that coupling the tetrapeptide to a short chain LAA enhances its delivery across human epidermis.

Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hvdrophobic peptide dramatically enhances in vitro and in vivo function

A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99% +/- 15.69 S.D. observed with CP, to 12.08% +/- 3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95% +/- 14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar. conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides. (c) 2006 Elsevier B.V. All rights reserved.

Self-condensation of a thiazole-peptide bearing a 21-membered loop into a library of giant macrocycles with multiple orthogonal loops

Tetrapeptide analogue H-[Glu-Ser-Lys(Thz)]-OH, containing a turn-inducing thiazole constraint, was used as a template to produce a 21-membered structurally characterized loop by linking Glu and Lys side chains with a Val-Ile dipeptide. This template was oligomerized in one pot to a library (cyclo-[1](n), n = 2-10) of giant symmetrical macrocycles (up to 120-membered rings), fused to 2-10 appended loops that were carried intact through multiple oligomerization (chain extension) and cyclization (chain terminating) reactions of the template. A three-dimensional solution structure for cyclo-[1](3) shows all three appended loops projecting from the same face of the macrocycle. This is a promising approach to separating pepticle motifs over large distances.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Peptide conjugate hydrogelators

Molecular gelators are currently receiving a great deal of attention. These are small molecules which, under the appropriate conditions, assemble in solution to, in the majority of cases, give long fibrillar structures which entangle to form a three-dimensional network. This immobilises the solvent, resulting in a gel. Such gelators have potential application in a number of important areas from drug delivery to tissue engineering. Recently, the use of peptide-conjugates has become prevalent with oligopeptides (from as short as two amino acids in length) conjugated to a polymer, alkyl chain or aromatic group such as naphthalene or fluorenylmethoxycarbonyl (Fmoc) being shown to be effective molecular gelators. The field of gelation is extremely large; here we focus our attention on the use of these peptide-conjugates as molecular hydrogelators.

Peptide binding prediction for the human class II MHC allele HLA-DP2:a molecular docking approach

MHC class II proteins bind oligopeptide fragments derived from proteolysis of pathogen antigens, presenting them at the cell surface for recognition by CD4+ T cells. Human MHC class II alleles are grouped into three loci: HLA-DP, HLA-DQ and HLA-DR. In contrast to HLA-DR and HLA-DQ, HLA-DP proteins have not been studied extensively, as they have been viewed as less important in immune responses than DRs and DQs. However, it is now known that HLA-DP alleles are associated with many autoimmune diseases. Quite recently, the X-ray structure of the HLA-DP2 molecule (DPA*0103, DPB1*0201) in complex with a self-peptide derived from the HLA-DR a-chain has been determined. In the present study, we applied a validated molecular docking protocol to a library of 247 modelled peptide-DP2 complexes, seeking to assess the contribution made by each of the 20 naturally occurred amino acids at each of the nine binding core peptide positions and the four flanking residues (two on both sides).

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.

Polymer-peptide conjugate hydrogels:towards controlled drug delivery

Peptide-based materials exhibit remarkable supramolecular self-assembling behavior, owing to their overwhelming propensity to from hierarchical structures from a-helices and ß-sheets. Coupling a peptide sequence to a synthetic polymer chain allows greater control over the final physical properties of the supermolecular material. So-called ‘polymer-peptide conjugates’ can be used to create biocompatible hydrogels which are held together by reversible physical interactions. Potentially, the hydrogels can be loaded with aqueous-based drug molecules, which can be injected into targeted sites in the body if they can exhibit a gel-sol-gel transition under application and removal of a shear force. In this review, we introduce this topic to readers new to the field of polymer-peptide conjugates, discussing common synthetic strategies and their self-assembling behavior. The lack of examples of actual drug delivery applications from polymer-peptide conjugates is highlighted in an attempt to incite progress in this area.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented).

Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background - MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results - A large dataset comprising MHC-peptide structural complexes was created by re-modelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion - The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.