Synthesis and antiviral evaluation of ribavirin congeners containing a hexitol moiety.

Several ribavirin congeners containing a hexitol moiety were prepared via ring opening of two different epoxides with the methylcarboxylate ester of triazole and further elaboration. Unfortunately, none of the newly synthesized compounds displayed appreciable antiviral activity.

Chemical incorporation of 1-methyladenosine, minor tRNA component, into oligonucleotides.

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.

Methylated hexitol nucleic acids, towards congeners with improved antisense potential.

In an effort to further improve the hybridisation potential of anhydro-hexitol nucleoside analogues, the 1'-methoxyl and 3'-methoxyl substituents were introduced and evaluated for their antisense potential. In view of the selectivity of pairing with RNA, especially the introduction of a 3'-O-alkyl moiety deserves further study.

Mechanism of simmondsin decomposition during sodium hydroxide treatment.

Jojoba seed meal shows appetite-suppressing activity due to the presence of simmondsin. This pharmacological activity disappears with treatment of the meal with sodium hydroxide. To elucidate this mechanism of inactivation, the reaction of simmondsin in 1 N NaOH at 20 degrees C was monitored as a function of time. The end products of the reaction as well as intermediates were isolated and identified. The half-life of simmondsin was approximately 60 min with d-glucose and 2-hydroxy-3-methoxyphenylacetonitrile as reaction end products. The reaction mechanism could be elucidated by the isolation of isosimmondsin and a simmondsin lactone derivative. Those compounds were isolated and purified by a combination of column chromatography and HPLC and identified mainly by HRMS and NMR spectroscopy.

Improved hybridisation potential of oligonucleotides comprising O-methylated anhydrohexitol nucleoside congeners.

The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.

Identification of 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin and pinitol alpha-D-galactosides in jojoba seed meal (Simmondsia chinensis).

The isolation and identification of two pinitol alpha-D-galactosides from jojoba meal are described. The products were isolated by a combination of preparative HPLC on silica gel and TLC on amino silica gel and were identified by MS, NMR spectroscopy, and chemical derivatization as 5-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 5-alpha-D-galactopyranosyl-D-pinitol and 2-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 2-alpha-D-galactopyranosyl-D-pinitol. The same preparative HPLC method on silica gel allowed a new simmondsin derivative to be isolated and identified as 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin mainly by NMR spectroscopy and high-resolution mass spectrometry.

Isolation and structural characterization of colistin components.

Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrile-sodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) - water (16:50:34, % v/v/v) was carried out at a flow rate of 4.0 ml min(-1). Six colistin components were isolated and characterized using 1H and 13C NMR. The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1, except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin E1 with 7-methylnonanoic acid as the fatty acid moiety.

Isolation and structural characterization of polymyxin B components.

Polymyxin B is a peptide antibiotic complex present as sulphate. The components were separated preparatively on a poly(styrene-divinylbenzene) (PLRP-S), 1000 A, 8 microm, 250 x 12.5 mm I.D. stationary phase maintained at 60 degrees C and using 215 nm detection. Elution was carried out with acetonitrile-sodium sulphate solution (0.7%, m/v; pH adjusted to 2.5 with trifluoroacetic acid)-water (18:50:32, v/v) at a flow-rate of 4.0 ml/min. Seven polymyxin B components were isolated and characterized using 1H and 13C NMR. The molecular masses were confirmed by mass spectrometry. The structures of two components were determined for the first time. Polymyxins B5 and B6 were identified as having the same composition as polymyxin B1 except that the fatty acid moiety was nonanoic acid and 3-hydroxy-6-methyloctanoic acid, respectively.

Physical stabilisation of amorphous ketoconazole in solid dispersions with polyvinylpyrrolidone K25.

The glass forming properties of ketoconazole were investigated using differential scanning calorimetry (DSC), by quench cooling liquid ketoconazole from T(m)+10 to 273.1 K, followed by subsequent heating at 5 K/min to T(m)+10 K. It was shown that liquid ketoconazole forms a glass which did not recrystallise following reheating, indicating its stability; T(g) was found to be 317.5+/-0.3 K. However, the presence of a small amount of crystalline ketoconazole was able to convert the amorphous drug back to the crystalline state: the addition of only 4.1% (w/w) of crystalline material converted 77.1% of the glass back to the crystalline state, and this value increased as the amount of added crystals increased. PVP K25 was found to be highly effective in the prevention of such recrystallisation, but only if the amorphous drug was formulated in a solid dispersion, since physical mixing of amorphous ketoconazole with the polymer resulted in recrystallisation of the former compound. Storage of the solid dispersions for 30 days at 298.1 K (both 0 and 52% RH) in the presence or absence of crystals did not result in recrystallisation of the amorphous drug. Solid dispersions formed compatible blends as one single T(g) was observed, which gradually increased with increasing amounts of PVP K25, indicating the anti-plasticising property of the polymer. The values of T(g) followed the Gordon-Taylor equation, indicating no significant deviation from ideality and suggesting the absence of strong and specific drug-polymer interactions, which was further confirmed with 13C NMR and FT-IR. It can be concluded therefore that the physical mechanism of the protective effect is not caused by drug-polymer interactions but due to the polymer anti-plasticising effect, thereby increasing the viscosity of the binary system and decreasing the diffusion of drug molecules necessary to form a lattice.

Hybridization between "six-membered" nucleic acids: RNA as a universal information system.

Within the polyA:polyT recognition system, cross-pairing between several nucleic acids with a phosphorylated six-membered carbohydrate (mimic) as repeating unit in the backbone structure has been observed. All investigated nucleic acids (except for beta-homo-DNA) hybridize with RNA, leaving RNA as a versatile biopolymer for informational transfer. [reaction: see text]

Alpha-homo-DNA and RNA form a parallel oriented non-A, non-B-type double helical structure.

Cross-talking between nucleic acids is a prerequisite for information transfer. The absence of observed base pairing interactions between pyranose and furanose nucleic acids has excluded considering the former type as a (potential) direct precursor of contemporary RNA and DNA. We observed that alpha-pyranose oligonucleotides (alpha-homo-DNA) are able to hybridize with RNA and that both nucleic acid strands are parallel oriented. Hybrids between alpha-homo-DNA and DNA are less stable. During the synthesis of alpha-homo-DNA we observed extensive conversion of N6-benzoyl-5-methylcytosine into thymine under the usual deprotection conditions of oligonucleotide synthesis. Alpha-homo-DNA:RNA represents the first hybridization system between pyranose and furanose nucleic acids. The duplex formed between alpha-homo-DNA and RNA was investigated using CD, NMR spectroscopy, and molecular modeling. The general rule that orthogonal orientation of base pairs prevents hybridization is infringed. NMR experiments demonstrate that the base moieties of alpha-homo-DNA in its complex with RNA, are equatorially oriented and that the base moieties of the parallel RNA strand are pseudoaxially oriented. Modeling experiments demonstrate that the duplex formed is different from the classical A- or B-type double stranded DNA. We observed 15 base pairs in a full helical turn. The average interphosphate distance in the RNA strand is 6.2 A and in the alpha-homo-DNA strand is 6.9 A. The interstrand P-P distance is much larger than found in the typical A- and B-DNA. Most helical parameters are different from those of natural duplexes.

Solution structure of a HNA-RNA hybrid.

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis.

Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) methylates, in vitro, caffeoyl-CoA and 5-hydroxyferuloyl-CoA, two possible precursors in monolignol biosynthesis in vivo. To clarify the in vivo role of CCoAOMT in lignin biosynthesis, transgenic poplars with 10% residual CCoAOMT protein levels in the stem xylem were generated. Upon analysis of the xylem, the affected transgenic lines had a 12% reduced Klason lignin content, an 11% increased syringyl/guaiacyl ratio in the noncondensed lignin fraction, and an increase in lignin-attached p-hydroxybenzoate but otherwise a lignin composition similar to that of wild type. Stem xylem of the CCoAOMT-down-regulated lines had a pink-red coloration, which coincided with an enhanced fluorescence of mature vessel cell walls. The reduced production of CCoAOMT caused an accumulation of O(3)-beta-d-glucopyranosyl-caffeic acid, O(4)-beta-d-glucopyranosyl-vanillic acid, and O(4)-beta-d-glucopyranosyl-sinapic acid (GSA), as authenticated by (1)H NMR. Feeding experiments showed that O(3)-beta-d-glucopyranosyl-caffeic acid and GSA are storage or detoxification products of caffeic and sinapic acid, respectively. The observation that down-regulation of CCoAOMT decreases lignin amount whereas GSA accumulates to 10% of soluble phenolics indicates that endogenously produced sinapic acid is not a major precursor in syringyl lignin biosynthesis. Our in vivo results support the recently obtained in vitro enzymatic data that suggest that the route from caffeic acid to sinapic acid is not used for lignin biosynthesis.

Investigation of the kinetics of degradation of hexopyranosylated cytosine nucleosides using liquid chromatography.

Liquid chromatography was used to follow the degradation of hexopyranosylated cytosine nucleosides in buffers of acid, neutral and alkaline pH and of constant ionic strength. The compounds were found to degrade by hydrolysis to cytosine and/or by deamination to the corresponding uracil nucleosides. Degradation in acid is influenced by the number of sugar hydroxyl groups, presence of sugar double bonds and the type of anomer. Stability of some of the compounds was compared with that of related thymine nucleosides. Temperature studies support a unimolecular mechanism of hydrolysis at pH 1.22.

Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides.

Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.

Synthesis, biological activity, and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y(1) receptor ligands.

The structure-activity relationships of adenosine-3', 5'-bisphosphates as P2Y(1) receptor antagonists have been explored, revealing the potency-enhancing effects of the N(6)-methyl group and the ability to substitute the ribose moiety (Nandanan et al. J. Med. Chem. 1999, 42, 1625-1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering), non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity of each analogue at P2Y(1) receptors was characterized by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation elicited by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). Addition of the N(6)-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N(6)-methyl-2'-deoxyadenosine bisphosphate analogue was a pure P2Y(1) receptor antagonist and equipotent to the ribose analogue (MRS 2179). In the series of ring-constrained methanocarba derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle, the 6-NH(2) (N)-analogue was a pure agonist of EC(50) 155 nM and 86-fold more potent than the corresponding (S)-isomer. The 2-chloro-N(6)-methyl-(N)-methanocarba analogue was an antagonist of IC(50) 51.6 nM. Thus, the ribose ring (N)-conformation appeared to be favored in recognition at P2Y(1) receptors. A cyclobutyl analogue was an antagonist with IC(50) of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydrohexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-NH(2)) or antagonists (N(6)-methyl). A molecular model of the energy-minimized structures of the potent antagonists suggested that the two phosphate groups may occupy common regions. The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site of the previously reported P2Y(1) receptor model.

Synthesis and apoptogenic activity of fluorinated ceramide and dihydroceramide analogues.

Short-chain 3-fluoro-(dihydro)ceramide analogues are synthesized from L-serine using diethylaminosulfur trifluoride (DAST) as fluorinating agent. The apoptogenic activity of these compounds was measured in three different cell lines and compared with their hydroxylated counterparts.

Structure-activity relationship of short-chain sphingoid bases as inhibitors of sphingosine kinase.

Short-chain sphinganine analogues 8, 9, 18, and 19, as well as 3-fluoro-sphingosine analogues 25 and 26 were synthesized. Their potential as sphingosine kinase inhibitors was investigated, in combination with previously synthesized sphingosine and fluorinated sphinganine analogues.

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei.

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

Base pairing of anhydrohexitol nucleosides with 2,6-diaminopurine, 5-methylcytosine and uracil asbase moiety.

Hexitol nucleic acids (HNAs) with modified bases (5-methylcytosine, 2,6-diaminopurine or uracil) were synthesized. The introduction of the 5-methylcytosine base demonstrates that N -benzoylated 5-methylcytosyl-hexitol occurs as the imino tautomer. The base pairing systems (G:CMe, U:D, T:D and U:A) obey Watson-Crick rules. Substituting hT for hU, hCMefor hC and hD for hA generally leads to increased duplex stability. In a single case, replacement of hC by hCMedid not result in duplex stabilization. This sequence-specific effect could be explained by the geometry of the model duplex used for carrying out the thermal stability study. Generally, polypurine HNA sequences give more stable duplexes with their RNA complement than polypyrimidine HNA sequences. This observation supports the hypothesis that, besides changes in stacking pattern, the difference in conformational stress between purine and pyrimidine nucleosides may contribute to duplex stability. Introduction of hCMeand hD in HNA sequences further increases the potential of HNA to function as a steric blocking agent.