Goran Kragol

Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2.

Matix protein 2 (M2) is a transmembrane protein of influenza type A virus. It contains a 23 aa long ectodomain (M2e) that is highly conserved amongst human influenza type A viruses. M2e-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection. We attempted to induce M2e-specific protection with synthetic multiple antigen peptide (MAP) constructs that contained covalently linked M2e- and Th-determinant peptides. Mice, vaccinated twice by the intranasal (i.n.) route with adjuvanted M2e-MAPs exhibited significant resistance to virus replication in all sites of the respiratory tract. Compared to mice primed by two consecutive heterosubtypic infections, resistance was of similar strength in nasal and tracheal tissue but lower in pulmonary tissue. Importantly, the protection in M2e-MAP- and infection-immunized mice appeared to be mediated by distinct immune mechanisms. This suggests that stronger protection may be achievable by combining both protective activities.

Tumor-Cell-Targeted Methionine-enkephalin Analogues Containing Unnatural Amino Acids: Design, Synthesis, and in Vitro Antitumor Activity

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

Synthesis and alkali metal picrate extraction capabilities of novel cage-functionalized 17-crown-5 and 17-crown-6 ethers

The syntheses of three novel cage-functionalized 17-crown-5 and 17-crown-6 ethers, i.e., 10–12, are reported and the results of alkali metal picrate extraction experiments performed by using each of these crown ethers in turn as the extractant are described. Their respective relative extraction efficiencies toward alkali metal picrates were compared with the corresponding efficiencies of 15-crown-5 and benzo-15-crown-5 solutions at the same concentration. The extraction profile of 10 (17-crown-5) toward alkali metal picrates is similar to that of 15-crown-5; however, 10 displays 33% greater avidity toward Na+ picrate. The ability of 11 (benzo-17-crown-5) to extract Li+, Rb+, and Cs+ picrates is similar to that of benzo-15-crown-5. Crown ether 10 is ca. 3 times more effective than 11 toward extraction of K+ picrate. The alkali metal picrate extraction profile of 12 (17-crown-6) resembles that of 10; however, 12 shows enhanced avidity vis-a-vis 10 toward Na+, K+, Rb+, and Cs+ picrates.

Orthogonal solid-phase synthesis of tetramannosylated peptide constructs carrying three independent branched epitopes

Abstract The development of peptide-based drugs requires the chemical synthesis of systems as complex as they appear in nature. Most bioactive peptides have to be associated with co-activators and delivery or targeting domains, the synthesis of such complexes is far from trivial. In efforts to develop a prototype for a new generation of peptide vaccines, a peptide construct was prepared, using an alternating lysine and glycine backbone. A 24-mer major antigen corresponding to the M2 protein of influenza virus, and two shorter T-cell epitopes derived from the hemagglutinin were co-synthesized onto the side chains of the first three lysines. To help the delivery of the constructs inside the antigen presenting cells via the multimeric cell surface mannose receptor, three additional lysines were decorated with four mannosylated serine residues. The synthesis difficulty increased upon addition of the glycoamino acids and alternating the peptide and glycoamino acid branches. The successful solid-phase synthesis of the constructs proceeded with the use of a combination of three quasi-orthogonally removable amino protecting groups and a robust activation strategy. These multi-glycosylated constructs represent some of the most complex synthetic peptides to date, and will be used to study the entire process of antigen delivery, presentation and immunogenicity.

Design, synthesis and cation-binding properties of novel adamantane- and 2-oxaadamantane-containing crown ethers

Abstract The synthesis of a series of adamantane- and 2-oxaadamantane-functionalized crown ethers 1–7 is described. Alkali metal picrate extraction profiles have been determined for these novel ionophores. The ability of crown ethers 1–7 to extract the alkali metal picrates was compared with that of benzo-15-crown-5 and 18-crown-6. Also, Na+- and K+-transport, the ability of ionophores l–3 to transport Na+- and K+ across a bulk liquid membrane was measured. The results of alkali metal cation extraction experiments showed that the complexation properties of crown ethers 1 and 2 are comparable to that of benzo-15-crown-5 and 18-crown-6, respectively. Crown ether 3 showed enhanced extractability for all cations but lower selectivity compared to 18-crown-6. However, adamantano-crown ethers 4–7 showed almost negligible extraction capability toward any of the alkali cations. The observed differences among the complexation abilities of the ionophores 1–3 and 4–7 are rationalized on the basis of the results of a molecular modeling study of their corresponding K+ complexes.

Stabilization of a K+-(bis-Cage-Annulated 20-Crown-6) Complex by Bidentate Picrate

Bis-cage-annulated 18-crown-6 and 20-crown-6 macrocyclic ethers (i.e., 1 and 2, respectively) have been synthesized, and their alkali metal picrate extraction profiles have been determined. Host system 1 proved to be a significantly more avid alkali metal cation complexant than 2 and somewhat more avid than 18-crown-6. Both 1 and 18-crown-6 display modest selectivity toward K+ and Rb+. A stable host–guest complex was prepared by slow evaporation of a CH2Cl2–hexane solution of an equimolar mixture of 2 and potassium picrate. The X-ray crystal structure of this complex reveals that picrate anion functions as a bidentate ligand therein. The gas-phase interaction energy between the 2 ⋅ K+ complex and picrate anion was calculated to be ca. −64.9 kcal mol−1, thereby indicating that participation of picrate anion as an additional bidentate ligand results in significant stabilization of complex 10.

Fluorescently labeled macrolides as a tool for monitoring cellular and tissue distribution of azithromycin

Exceptional therapeutic effects of macrolides in treating various infections and inflammatory conditions can be significantly contributed to their unique pharmacokinetic properties. Macrolides accumulate in cells and tissues, with concentrations usually 10 to more than 100 times higher of those measured in plasma. Intracellular distribution of macrolides has so far been examined using extensive subcellular fractionation techniques, radiolabeled compounds and conventional pharmacokinetic methods. In this study we evaluated four fluorescently labeled macrolides on their applicability to monitor azithromycin distribution in vitro and in vivo. 9-Deoxo-9a-{3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl}-9a-aza-9a-homoerythromycin A (9a-NBD-azithromycin) was selected as a compound with most similar cellular pharmacokinetics to azithromycin. 9a-NBD-azithromycin demonstrated antimicrobial properties comparable to azithromycin, displayed the same biological activity profile in LPS-stimulated J774A.1 murine macrophage cells and, even though it accumulated in cells almost 50% more than azithromycin, it showed same rate of retention. Identical to azithromycin, 9a-NBD-azithromycin was localized in lysosomes of J774A.1 cells. Two hours after 9a-NBD-azithromycin was administered intraperitonally to mice, a strong fluorescent signal was located in kidneys and liver and slightly weaker in the spleen. In kidneys, the signal was concentrated in tubuli, and glomeruli were negative. Patchy florescence in hepatocytes supports lysosomal cellular localization. Weaker staining of white pulp compared to red pulp of spleen is in agreement with lower accumulation of azithromycin in lymphocytes compared to other cell types present. We conclude that 9a-NBD-azithromycin can be used as a fluorescent analog of azithromycin to visualize its distribution in in vitro systems, and is also suitable for in vivo studies.

Novel desosamine-modified 14- and 15-membered macrolides without antibacterial activity

Novel modifications of the desosamine sugar of 14- and 15-membered antibacterial macrolides, in which the desosamine was fused with N-substituted-1,3-oxazolidin-2-ones, were developed in order to completely suppress antibacterial activity and make them promising agents for other biological targets. The synthesis of such bicyclic desosamine derivatives, especially 1,3-oxazolidin-2-one formation, was optimized and conducted under mild conditions without a need for protection/deprotection steps for other functional groups. A focused series of novel desosamine-modified macrolide derivatives was prepared and their antibacterial activities tested. It was shown that these macrolide derivatives do not possess any residual antibacterial activity.

Molecular structure of bis[(1,3)2-oxaadamantano]-18-crown-6 and its potassium picrato complex

Abstract The structure of bis[(1,3)2-oxaadamantano]-18-crown-6 (1) and its potassium picrato complex {[1-K]+(C6H2N3O7)−;} have been studied by means of NMR-spectroscopy, X-ray structure analysis and molecular mechanics calculations. In the gas phase and in the CDCl3 solution, in both 1 and [1-K]+ the most stable conformation of C2h symmetry is found. However, in the solid state 1 adopts crystallographic Ci symmetry while its potassium picrato complex exhibits C1 symmetry.

Impact of stereochemistry on the biological activity of novel oleandomycin derivatives.

A set of 8-methylene-, 8-methyl-, and 8-methyl-9-dihydro-oleandomycin derivatives having different combinations of stereochemistries at positions C-8 and/or C-9 have been prepared in a chemoselective and stereoselective manner and tested in vitro for antibacterial activity and inhibition of IL-6 production. Configurations of the stereocenters at C-8 and C-9 were determined using 2D NMR techniques. We have shown that change of stereochemistry at these positions can exert a major influence on antibacterial activity as well as IL-6 inhibition, providing novel macrolide derivatives with diminished antibacterial and potent anti-inflammatory activity. In addition, the anti-inflammatory activity observed in vitro was confirmed in an in vivo model of lipopolysaccharide-induced inflammation.