Paweł Sowiński

A Novel Nucleotide Found in Human Erythrocytes, 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, 1H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-β-d-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-β-d-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 ± 0.006 μm) in contrast with the high daily urine excretion of this compound (26.7 ± 18.2 μmol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 ± 0.321 μm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

Molecular mechanism of the enzymatic oxidation investigated for imidazoacridinone antitumor drug, C-1311.

The imidazoacridinone derivative, C-1311, is an antitumor agent that has been under phase I of clinical trial. The work presented here aims to elucidate the molecular mechanism of the enzymatic oxidative activation of this drug in such a model metabolic system, where the covalent binding to DNA was previously demonstrated. The oxidative activation of C-1311 was performed with HRP/H(2)O(2) and MPO/H(2)O(2) systems. The obtained final products of such transformations were separated and analysed by HPLC. The structures of the products were identified by means of ESI-MS and NMR. It was demonstrated that C-1311 was oxidised with HRP and MPO in the manner dependent on the drug:H(2)O(2) ratio and the drug was more susceptible to HRP oxidation than to MPO. Structural studies showed compounds C0 and C1 to be the result of dealkylation, which occurred in the amino groups of the side chain. The structures of C3 and C4 products were identified as dimers, whose monomers held the imidazoacridinone core. The activation of the imidazoacridinone ring system in position ortho to 8-hydroxyl group was necessary to form such dimers. We suggest that similar mechanism of C-1311 activation should occur in the presence of DNA when, instead of the dimer formation, the covalent binding to DNA, showed earlier for this drug, was formed. Since peroxidase-type enzymes are present in the cell nucleus of tumour cells the activation mechanisms of the C-1311 proposed here may be expected to take place in the cellular environment in vivo.

Isolation of xanthone and benzophenone derivatives from Cyclopia genistoides (L.) Vent. (honeybush) and their pro-apoptotic activity on synoviocytes from patients with rheumatoid arthritis.

A fast and efficient method for the isolation of the C-glucosidated xanthones mangiferin and isomangiferin from the South-African plant Cyclopia genistoides was developed for the first time. The procedure involved extraction, liquid-liquid partitioning with ethyl acetate and subsequent precipitation of mangiferin and isomangiferin from methanol and acetonitrile-water fractions, respectively. Additionally, two benzophenone derivatives: 3-C-β-glucosides of maclurin and iriflophenone, were isolated from C. genistoides extracts using semi-preparative HPLC. Apart from the above, the isolation procedure also yielded hesperidin and small amounts of luteolin. The structures of the compounds were determined by 1D and 2D NMR experiments and/or LC-DAD-ESI-MS. The selected Cyclopia constituents were screened for pro-apoptotic activity on TNF-α-stimulated synovial cells isolated from rheumatoid arthritis patients. The strongest effect, measured as percent of apoptotic cells, was recorded for isomangiferin (75%), followed by iriflophenone 3-C-β-glucoside (71%), hesperidin (67%) and mangiferin (65%). The results are encouraging for further studies on the use of the above compounds in the treatment of rheumatoid arthritis.

Micropropagation of Cyclopia genistoides, an endemic South African plant of economic importance.

An efficient micropropagation protocol of Cyclopia genistoides (L.) Vent., an indigenous South African shrub of economic importance, was established. In vitro shoot cultures were obtained from shoot tip fragments of sterile seedlings cultured on solid Schenk and Hildebrandt (SH) medium supplemented with 9.84 μM 6-(γ,γ-dimethylallylamino)purine (2iP) and 1.0 μM thidiazuron (TDZ). Maximum shoot multiplication rate [(8.2 ± 1.3) microshoots/explant)] was observed on this medium composition. Prior to rooting, the multiplied shoots were elongated for 60 days (two 30-days passages) on SH medium with one-half sucrose concentration, supplemented with 4.92 μM indole-3-butyric acid (IBA). The rooting of explants was only possible in the case of the elongated shoots. The highest root induction rate (54.8%) was achieved on solid SH medium with one-half sucrose and one-half potassium nitrate and ammonium nitrate concentration, respectively, supplemented with 28.54 μM indole-3-acetic acid (IAA) and 260.25 μM citric acid. The plantlets were acclimatized for 30 days in the glasshouse, with the use of peat/gravel/perlite substrate (1:1:1). The highest acclimatization rate (80%) was obtained for explants rooted with the use of IAA-supplemented medium. The phytochemical profile of the regenerated plants was similar to that of the reference intact plant material. HPLC analyses showed that C. genistoides plantlets obtained by the micropropagation procedure kept the ability to produce xanthones (mangiferin and isomangiferin) and the fl avanone hesperidin, characteristic of wild-growing shrubs.

The structure, including stereochemistry, of levorin A1

The constitution and stereostructure of levorin A1 1, an aromatic heptaeneantifungal antibiotic, was established on the basis of NMR studies, which contained DQFCOSY,ROESY, HSQC and HMBC experiments. Mycosamine moiety was used as an internalchiral probe to determine the absolute configuration of levorin A1 stereogenic centers: 13S,15R, 17S, 18R, 19S, 21R. The relative configuration of the remaining stereogenic centers wasassigned as follows: 36S*, 37R*, 38S*, 40S* and 41S*. The configuration at C-5 still remainsto be established. The geometry of the heptaene chromophore was defined as 22E, 24E, 26Z,28Z, 30E, 32E and 34E.

Synthesis, Chemical Characterization and Multiscale Biological Evaluation of a Dimeric-cRGD Peptide for Targeted Imaging of α V β 3 Integrin Activity

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker αVβ3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of αVβ3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for αVβ3 in HUVECs (Kd ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within αVβ3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of αVβ3 expression, both in vitro and in vivo.

Stereostructure of mycoheptin A2

The absolute configurations of all the stereogenic centers of the antibiotic mycoheptin A2 were established upon previously elaborated general procedure, consisting of DQF‐COSY, NOESY, ROESY, HSQC and HMBC experiments as major tools. The structure of mycoheptin A2 without stereochemistry of its aglycone has been reported before. Copyright

Intercalation complex of imidazoacridinone C-1311, a potential anticancer drug, with DNA helix d(CGATCG)2: stereostructural studies by 2D NMR spectroscopy

Imidazoacridinone C-1311 (Symadex®) is a powerful antitumor agent, which successfully made its way through the Phase I clinical trials and has been recommended for Phase II few a years ago. It has been shown experimentally that during the initial stage of its action C-1311 forms a relatively stable intercalation complex with DNA, yet it has shown no base-sequence specificity while binding to DNA. In this paper, the d(CGATCG)2:C-1311 intercalation complex has been studied by means of two-dimensional NMR spectroscopy, yielding a full assignment of the resonance lines observed in 1H NMR spectra. The observation of the intermolecular NOE contacts between C-1311 and DNA allowed locating the ligand between the guanine and adenine moieties. Formation of a symmetric complex was pointed out on the basis of the lack of a second set of the 1H resonances. The resulting stereostructure of the complex was then improved by means of molecular dynamics, using the CHARMM force field and GROMACS software. To this end, distance restraints derived from the NOESY cross-peak volumes were applied to the atomistic model of the d(CGATCG)2:C-1311 complex. Obtained results are in full agreement with biochemical data on the mechanism of action of C-1311, in particular with the previously postulated post-intercalation enzymatic activation of the studied drug.

Flavonoids from Pseudotsuga menziesii

1 Four O-acylated flavonol glycosides, new in the plant kingdom, were isolated from the needles of Pseudotsuga menziesii. Their structures were established by 1D and 2D NMR and MS data as: daglesioside I [kaempferol 3-O-[2’’,5’’-O-(4’’’,4IV-dihydroxy)-β- truxinoyl]-α-L-arabinofuranoside] (), daglesioside II [kaempferol 3-O-[2’’,5’’-O-(4’’’- hydroxy)-β-truxinoyl]-α-L-arabinofuranoside] (2), daglesioside III [kaempferol 3-O-[2’’,5’’- di-O-(E)-p-coumaroyl]-α-L-arabinofuranoside] (3), and daglesioside IV [kaempferol 3-O-[3’’,6’’-di-O-(E)-cinnamoyl]-β-D-glucopyranoside] (4). In addition, the known fl avonoids (E)-tiliroside, (E)-ditiliroside, astragalin (kaempferol 3-O-β-D-glucopyranoside), isorhamnetin, kaempferol, and quercetin were identifi ed. The cytotoxic activity of compounds 1 and 3 was evaluated towards the HL-60, HeLa, and MDA-MB468 cell lines.

Inactivation of glucosamine-6-phosphate synthase by N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid.

N3‐Oxoacyl derivatives of L‐2,3‐diaminopropanoic acid 1–4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine‐6‐phosphate (GlcN‐6‐P) synthase in a time‐ and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme–ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV–visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L‐cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N‐terminal fragment of GlcN‐6‐P synthase. On the other hand, the reaction of a double‐bond‐containing compound, 3, with CEE, CGIF and GlcN‐6‐P synthase led to the formation of a CS bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.