Peptide and glycopeptide dendrimers. Part II

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Solid phase synthesis of glycopeptide dendrimers with Tn antigenic structure and their biological activities. Part I

Multiple antigenic peptides containing dimeric Tn antigen [Ac‐(Tn)₂‐γ‐Abu]₄‐(Lys‐X)₂‐Lys‐β‐Ala ( V : X=0; VIII : X=γ‐Abu) and [Ac‐(Tn)₂‐γ‐Abu]₈‐(Lys‐X)₄‐(Lys‐X)₂‐Lys‐β‐Ala ( XI : X=0; XIV : X=γ‐Abu), immobilized on biocompatible Tenta Gel S NH₂ support were prepared by SPPS. Rosetting tests of V , VIII , XI and XIV showed positive reactions with anti‐Tn (DAKO) and Tn+ erythrocytes, with anti‐Tn/A (BRIC 66) and Tn+ and A erythrocytes, other combinations were negative. In all the animals immunized with XIV , we found a remarkable increase in the level of anti‐Tn (titre 2000–64 000, score 105–167) and no change of anti‐A levels (titre 8, score 13–17). Neither non‐immune nor immune sera showed any reactivity with T⁺, Cad⁺ and blood group O erythrocytes. Immunized mice did not exhibit any sign of adverse reaction to the administered conjugates. Biological activities were correlated with molecular modelling and molecular dynamic calculations. The biological activities of these synthetic Tn antigen conjugates (good availability for the immunological interactions, highly specific immunogenicity, good biological tolerance) together with their precise chemical characterization seem to be a promising approach to preparation of anti‐tumour vaccine and affinity purification of anti‐Tn antibodies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

Glycopeptide dendrimers. Part I.

Glycopeptide dendrimers are branched structures containing both carbohydrates and peptides. Various classes of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to glycopeptide dendrimers, namely multiple antigen glycopeptides (MAGs). In MAGs, the core, branches or both are composed of amino acids or peptides. Other classes of glycodendrimers (PAMAM, polypropylene imine, cyclodextrin, calixarene, etc.) are mentioned too, but to a smaller extent. Their syntheses, physicochemical properties and biological activities are given with many examples. Glycopeptide dendrimers can be used as inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, for studying of recognition processes, diagnostics, imaging and contrast agents, mimetics, for complexation of different cationts, as site-specific molecular delivery systems, for therapeutic purposes, as immunodiagnostics and in drug design. Biomedical applications of glycopeptide dendrimers as drug and gene delivery systems are also given.     

The effects of structural analogs of putrescine on proliferation,morphology and karyotype of glioblastoma cells in culture

Summary— In a previous study, we identified regions on the surface of tumor cells which act as acceptor sites for putrescine (Put) and studied the competition between structural analogs of Put (N,N′-tetramethyl-α,ω-diaminoalkanes) and Put bound to latex microspheres. A chain of four to seven carbons was necessary for inhibition of Put-latex binding to the cell surface of human glioblastoma (U251) cells. We show here that under the experimental conditions, N,N′-tetramethyl-1,4-butanediamine and N,N′-tetramethyl-1,7-heptanediamine exhibit an antitumor effect. In a first step (1–48 h after treatment), cells exposed to these compounds show large intracellular vacuoles. We failed to detect any acid phosphatase activity in these intracellular structures revealing that they were not lysosomes. Electron microscopy observations argue for the conclusion that these vacuoles are an hypertrophy of the endoplasmic reticulum (ER) and/or of the Golgi vesicles. Our hypothesis is that this typical effect of the analogs reveals that ER could be a physiological target of endogenous polyamines. At a later stage (6 days after treatment), the cells undergo morphological and biochemical changes: thin and long expansions characterize the cells and the GFA protein is overexpressed. Correlated to both these effects, karyotypic modifications are found in chromosomes 3 and 6. These changes evoke a differentiation of the treated cells. The work provides evidence that N-methylated polyamine analogs taking the place of endogenous putrescine demonstrate a hopeful antitumor effect.     

Glycopeptide dendrimers. Part II.

Glycopeptide dendrimers are regularly branched structures containing both carbohydrates and peptides. Various types of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. This Part II (for Part I see JeZek J, et al., J. Pept. Sci. 2008; 14: 2-43) covers linear oligomers with variable valency (brush dendrimers, comb dendrimers), sequential oligopeptide carriers SOCn-I and SOCn-II, chitosan-based dendrimers, and brush dendrimers. Other types of glycopeptide dendrimers are self-immolative dendrimers (cascade release dendrimers, domino dendrimers), dendrimers containing omega-amino acids (Gly, beta-Ala, gamma-Abu and epsilon-aminohexanoic acid), etc. Microwave-assisted synthesis of dendrimers and libraries of glycopeptides and glycopeptide dendrimers are also included. Characterization of dendrimers by electromigration methods, mass spectrometry, and time-resolved and nonlinear optical spectroscopy, etc. plays an important role in purity assessment and structure characterization. Physicochemical properties of dendrimers including chirality are given. Stability of dendrimers, their biocompatibility and toxicity are reviewed. Finally, biomedical applications of dendrimers including imaging agents (contrast agents), site-specific drug delivery systems, artificial viruses, synthetic antibacterial, antiviral, and anticancer vaccines, inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, etc. are given. Glycopeptide dendrimers were used also for studying recognition processes, as diagnostics and mimetics, for complexation of different cations, for therapeutic purposes, as immunodiagnostics, and in drug design.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

Glycopeptide dendrimers, part III: a review. Use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases.

Glycopeptide dendrimers containing different types of tumor associated-carbohydrate antigens (T(N), TF, sialyl-T(N), sialyl-TF, sialyl-Le(x), sialyl-Le(a) etc.) were used in diagnosis and therapy of different sorts of cancer. These dendrimeric structures with incorporated T-cell epitopes and adjuvants can be used as antitumor vaccines. Best results were obtained with multiantigenic vaccines, containing, e.g. five or six different TAAs. The topic of TAAs and their dendrimeric forms at molecular level are reviewed, including structure, syntheses, and biological activities. Use of glycopeptide dendrimers as antiviral vaccines against HIV and influenza is also described. Their syntheses, physico-chemical properties, and biological activities are given with many examples.     

Synthesis and binding potencies of cyclohexapeptide somatostatin analogs containing naphthylalanine and arylalkyl peptoid residues

We report the synthesis, binding affinities to the recombinant human somatostatin receptors, and structure‐activity relationship studies of compounds related to the cyclic hexapeptide, c‐[Pro⁶‐Phe⁷‐D‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹], L‐363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with the arylalkyl peptoid residues Nphe (N‐benzylglycine), (S)βMeNphe [(S)‐N‐[(α‐methyl)benzyl]glycine] or (R)βMeNphe [(R)‐N‐[(α‐methyl)benzyl]glycine] and l ‐1‐naphthylalanine is incorporated into either position 7 or 11 of the parent compound. The synthesis and binding data of the Nnal⁶ ([N‐naphthylmethyl]glycine) analog of L‐363,301 is also reported. The incorporation of the Nnal residue into position 6 of L‐363,301 resulted in an analog with weaker binding affinities to all hsst receptors but enhanced selectivity towards the hsst2 receptor compared with the parent compound. The other compounds bind effectively to the hsst2 receptor but show some variations in the binding to the hsst3 and hsst5 receptors resulting in different ratios of binding affinities to the hsst5 and hsst2 or hsst3 and hsst2, respectively. The incorporation of the Nphe residue into position 6 and the Nal residue into position 7 of L‐363,301 led to a compound which binds potently to the hsst2 and has increased selectivity towards this receptor (weaker binding to hsst3 and hsst5 receptors) compared with the parent compound. The analogs with β‐methyl chiral substitutions in the aromatic peptoid side chain and Nal in position 7 or 11 bind effectively to the hsst2 and hsst5 receptors. They exhibit similar ratios of binding affinities to the hsst5 and hsst2 receptors as observed for L‐363,301. There are however minor differences in binding to the hsst3 receptor among these analogs. These studies allow us to investigate the influence of additional hydrophobic groups on the binding activity to the isolated human somatostatin receptors and the results are important for the design of other somatostatin analogs. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of P450 inhibition and induction on the olfactory toxicity of β, β′-iminodipropionitrile (IDPN) in the rat

In addition to the neurotoxic effects of β, β′-iminodipropionitrile (IDPN) which have been previously reported by other investigators, the olfactory toxicity of this compound has recently been uncovered in this laboratory. Due to the apparently conflicting observations that the IDPN-induced lesion in the olfactory mucosa is very focal in nature (suggesting site-specific activation) and the observation by other investigators that the behavioral effects of IDPN appear to be due to the parent compound, we initiated studies into the possible role of the cytochrome P450 enzymes in the olfactory toxicity of IDPN. Immunohistochemical studies with antibodies raised against several different P450 isoforms revealed good correlation between IDPN-induced olfactory mucosal degeneration and the localization of a protein immunoreacting with an antibody to P450 2E1. Enzymatic studies revealed that there is approximately fivefold more ρ-nitrophenol hydroxylation activity in the olfactory mucosa than in the liver on a per milligram microsomal protein basis. Administration of 1% acetone in the drinking water increased the levels of olfactory mucosal 2E1, and the increase in enzyme levels corresponded to increased olfactory toxicity of IDPN; inhibition of P450 activities with either metyrapone or carbon tetrachloride eliminated or significantly decreased the olfactory toxicity of IDPN, respectively. These studies suggest a role for cytochrome P450, specifically the 2E1 isoform, in the activation of IDPN within the nasal mucosa.     

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN‐LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d ‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d ‐glucan.     

Synthesis and alkylation of aza‐glycinyl dipeptide building blocks

Aza‐glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza‐glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by‐products. Employing N,N′‐disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza‐glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza‐glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza‐propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross‐couplings, dipolar cycloadditions and intramolecular exo‐dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis, structural characterization and in vitro cytotoxicity and anti-bacterial activity of some copper(I) complexes with N,N′-disubstituted thioureas

A series of copper(I) complexes of N,N′-disubstituted thioureas, [C₆H₅CONHCSNHR]Cu(I)Cl where R = C₆H₅ (1a), 2-ClC₆H₄ (2a), 3-ClC₆H₄ (3a), 4-ClC₆H₄ (4a), 2,3-Cl₂C₆H₃ (5a), 2,4-Cl₂C₆H₃ (6a), 2,5-Cl₂C₆H₃ (7a), 2,6-Cl₂C₆H₃ (8a), 3,4-Cl₂C₆H₃ (9a) and 3,5-Cl₂C₆H₃ (10a) have been synthesized. These complexes (1a–10a) have been characterized by elemental analyses, IR, ¹H and ¹³C NMR spectroscopy, cyclic voltammetry and single crystal XRD for 1a and 8a, and for ligand 7. The X-ray crystal structures reveal that the complexes 1a and 8a are mononuclear in the solid state in which the copper atoms adopt a distorted tetrahedral geometry. In both the cases, the neutral N,N′-disubstituted thiourea ligands have been coordinated to the Cu(I) through the sulphur atom in a terminal mode. The complexes have been screened for their in vitro cytotoxic activity in human cell lines carcinomas A498 (Renal), EVSA-T (Breast), H226 (Lung), IGROV (Ovarian), M19 (Melanoma-Skin), MCF-7 (Breast) and WIDR (Colon). They show a moderate cytotoxicity against these seven human cancer cell lines comparable to that of the less active standard chemotherapeutic drugs used for comparison. They were also screened for their anti-bacterial activity and were found less active than the standard drug Imipenem.     

Isolation and characterization of α‐(1→3)‐glucan‐degrading bacteria from the gut of Diaperis boleti feeding on Laetiporus sulphureus

Bacteria degrading α‐(1→3)‐glucan were sought in the gut of fungivorous insects feeding on fruiting bodies of a polypore fungus Laetiporus sulphureus, which are rich in this polymer. One isolate, from Diaperis boleti, was selected in an enrichment culture in the glucan‐containing medium. The bacterium was identified as Paenibacillus sp. based on the results of the ribosomal DNA analysis. The Paenibacillus showed enzyme activity of 4.97 mU/cm³ and effectively degraded fungal α‐(1→3)‐glucan, releasing nigerooligosaccharides and a trace amount of glucose. This strain is the first reported α‐(1→3)‐glucan‐degrading microorganism in the gut microbiome of insects inhabiting fruiting bodies of polypore fungi.     

Design and validation of a corneal bioreactor

Mechanical strain is an important signal that influences the behavior and properties of cells in a wide variety of tissues. Physiologically similar mechanical strain can revert cultured cells to a more normal phenotype. Here, we have demonstrated that 3% equibiaxial (EB) and uniaxial strains confer favorable protein expression in cultured rabbit corneal fibroblasts (RCFs), with approximately 35% and 65% reduction in expression of α‐smooth muscle actin (α‐SMA), respectively. We have designed a novel bioreactor that is capable of imparting up to 7% EB strain and up to 6% EB strain using a cornea‐shaped post. Additional features of the bioreactor include the application of shear stress to cells in culture and the ability to image cells using optical coherence microscopy (OCM) without being removed from the system. Biotechnol. Bioeng. 2012; 109: 3189–3198. © 2012 Wiley Periodicals, Inc.