Synthesis of biotinylated muramyl tripeptides with NOD2-stimulating activity

Muramyl di- and tri-peptides are putative activators of the innate immune system through stimulation of the NOD2 receptor. To provide tools for the clarification of the mechanism of this activation we isolated different UDP-muramyl tripeptides (Lys- and DAP-type) from bacteria and used them to synthesize biotinylated derivatives. All biotinylated compounds retained their ability to activate NOD2 in a cell-based test system and are therefore suitable for binding studies aimed at identifying the appropriate pattern recognition receptor(s).     

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.     

Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Synthesis of biologically active canine CCK-58

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity.     

Synthesis of biologically active transforming growth factor alpha

A 50-amino acid residue transforming growth factor, type alpha (TGF alpha), secreted in culture by feline-sarcoma-virus-transformed rat embryo fibroblasts, was synthesized by an improved stepwise solid-phase method with an overall yield of 31%. A deprotection strategy based on the SN2 mechanism using either a low concentration of HF or CF3SO3H-CF3CO2H in dimethylsulfide was employed to remove most of the benzyl-derived protecting groups. The more acid resistant protecting groups of Cys and Arg were removed by the SN2 condition using a high concentration of HF. Synthetic TGF alpha was purified to homogeneity in three steps. Synthetic and natural TGF alpha were indistinguishable from each other in HPLC and in different assays, including the assay for anchorage-independent growth of normal rat kidney fibroblasts in soft agar, binding, and stimulating to epidermal growth factor (EGF)-receptor protein kinase. Furthermore, synthetic TGF alpha showed similar biological activities when compared with EGF in these assays. Thus, the chemical synthesis of TGF alpha provided convincing evidence that TGF alpha is functionally related to EGF and is one of the active principles required for cellular transformation.     

Synthesis, mass spectral characterization and immunoadjuvant activity of some novel lipophilic derivatives of muramyl dipeptides

Some novel lipophilic derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) have been prepared and rigorously evaluated by spectroscopic means. Fast atom bombardment and field desorption mass spectrometry provided information about both molecular weight and structural detail. The new MDP derivatives have been tested in guinea pigs for immunoadjuvant activity using egg albumin as the model antigen. Amongst these derivatives, 6-O-[3-(5-cholesten-3 beta-yloxycarbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (CSMDP), 6-O-[3-1,2-dipalmitoyl-sn-glycero-3-carbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (GSMDP) and N-palmitoyl muramyl-L-alanyl-D-isoglutamine (PMDP) possessed significantly better activity than MDP, as judged by the antigen-specific antibody and delayed hypersensitivity responses in the immunized animals. In addition, CSMDP was found to induce strong delayed hypersensitivity response even in saline. These three active compounds were also tested for their pyrogenic response in rabbits, and were found to be lesser pyrogenic than MDP. Some of these MDP derivatives hold promise as adjuvants in immunization.     

Synthesis of two biologically active fluorescent probes of thymopentin

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.     

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.     

Synthesis and characterization of biologically active recombinant elk and horse FSH

The objective of this investigation was to clone and express the elk and horse common α-subunit and FSH β-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1^® and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.     

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.     

Structure-activity relationship for FR901464: a versatile method for the conversion and preparation of biologically active biotinylated probes

The structure-activity relationship for FR901464, a potent cell-cycle inhibitor, was examined by synthesizing its analogs. A versatile method for converting FR901464 was devised. This method made it possible to synthesize biologically active FR901464-biotin conjugates which could be used to isolate the binding proteins.     

Rhamnogalacturonan-II--a biologically active fragment

Rhamnogalacturonan-II inhibited the uptake of [¹⁴C]leucine and,consequently, the incorporation of [¹⁴C]leucine into acid-precipitableproteins by suspension-cultured tomato cells. Fractionationof rhamnogalacturonan-II showed that the lower molecular componentswere the most effective. KDO and apiose, both constituents ofrhamnogalacturonan-II, also inhibited [¹⁴C]leucine incorporationweakly, suggesting that these sugar residues may be an integralrequirement for the biological activity of rhamnogalacturonan-II.The incorporation of [¹⁴C]glutamate and [¹⁴C]histidine, andto a lesser extent [¹⁴C]proline and [¹⁴C]arginine, was alsoinhibited by rhamnogalacturonan-II; the incorporation of [¹⁴C]tyrosineand [¹⁴C]phenylalanine was little affected. This suggests thatrhamnogalacturonan-II exerts its effect by acting on certainmembrane transport systems. Key words: Rhamnogalacturonan-II, inhibition, protein synthesis, amino acid incorporation     

Synthesis of biologically active steroid derivatives by the utility of Lawesson's reagent

Steroid derivatives V, VI, VII and VIII reacted with Lawesson's reagent (LR) to produce spiro-oxazaphosphole-4',17-androstene derivative XI, diazaphospholoandrostane XIV and the thionated derivatives XVI and XVII, respectively. The structures of the new compounds were confirmed by analytical and spectroscopic evidence. A mechanism accounting for the formation of the new compounds was given. The in vitro antimicrobial activity of the new compounds were tested.     

Synthesis of biologically active copper oxide nanoparticles as promising novel antibacterial-antibiofilm agents

Abstract

In this study, we aimed to synthesize copper oxide nanoparticles (CuONPs) mediated by plant extract in an environmentally friendly way and to reveal their potential biological activities. Here we synthesized CuONPs by using different concentrations of aqueous leaf extract of Thymbra spicata at 80?°C to obtain Ts1CuONPs and Ts2CuONPs. Biosynthesized nanoparticles were characterized by using UV-Vis, AFM, FTIR, SEM-EDS, TEM, DLS and zeta potential analysis. The antibacterial activity of the nanoparticles was determined by calculation of the inhibition zone and minimum inhibitory concentration against selected bacterial strains. Moreover, the antioxidant activity of the as-synthesized nanoparticles was evaluated based on DPPH radical scavenging activity. The results indicate that the as-synthesized NPs have an average size of 26.8 and 21?nm for Ts1CuONPs and Ts2CuONPs, respectively. The formed CuONPs have more antibacterial action on gram-positive bacteria compared to gram-negative bacteria. In addition, CuONPs demonstrated good inhibition activity against biofilm formation of Staphylococcus aureus (S. aureus). Furthermore, the results showed that the smaller size of the CuONPs caused the higher cytotoxicity on L929 mouse fibroblast cells. The as-synthesized CuONPs exhibit antibacterial and antibiofilm potential against S. aureus, indicating that they may be attractive candidates to use in future therapeutic applications.     

Synthesis of Pro-XylaneTM: A new biologically active C-glycoside in aqueous media

The scope and limitation of Lubineau’s reaction were evaluated for the synthesis of C-glycosides (compounds 1–13). Further transformation of side chain carbonyl was also achieved (compounds 16–23). Optimization of these two steps was investigated in xylose case. Some of the compounds were shown to stimulate sulfated glycosaminoglycans (GAGs) synthesis. Compound 20 (called Pro-Xylane^TM) was identified as the best activator of GAGs biosynthesis. Pro-Xylane^TM was developed using environmentally friendly conditions relevant to ‘Green-Chemistry’ principles and launched on the market in September 2006. This compound is the first example of ‘Green’ chemical used in cosmetic.     

Synthesis of biotinylated OSW-1

OSW-1 is a highly potent anticancer natural saponin with an unknown mode of action. To determine its cellular target(s) biotinylated OSW-1 was successfully synthesized in nine steps.