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Biomedical subjects

J Rozenski

Publications and source records attributed to J Rozenski.

16 recordsLinked to original sources
PubMed1999

Determination of nearest neighbors in nucleic acids by mass spectrometry.

The identification of nearest-neighbor residues in nucleic acids provides useful constraints on establishment of base composition and sequence and is potentially applicable to a range of structural problems involving synthetic and natural polynucleotides. A new approach to this problem using electrospray ionization tandem mass spectrometry is based on measurement of precursor-product relationships derived from small fragment ions produced in the high-pressure ionization ("nozzle-skimmer") region of the instrument. Measured mass values of dinucleotide or other fragments, which give rise to mononucleotide ions N formed in the collision cell and transmitted by the second mass analyzer, establish the identities of residues adjacent to N. The technique is applicable to RNA and DNA, whether modified, or not, and is demonstrated using modified residues in nucleic acids up to the size of intact tRNA (76-mer). By monitoring of selected ion reaction channels, the method has been extended to LC/MS and to nearest-neighbor determinations directly in oligonucleotide mixtures.

DNA↗
PubMed1999

The RNA Modification Database: 1999 update.

The RNA Modification Database (http://medlib.med.utah.edu/RNAmods/) provides a comprehensive listing of naturally modified nucleosides in RNA. Each file includes: chemical structure; common name and symbol; type(s) of RNA in which found and corresponding phylogenetic distribution; Chemical s registry number and index name; and initial literature citations for structure characterization and chemical synthesis. New features include capability to search database files by name or substructural features, modifications in tmRNA, and links to related data and sites.

Archaea↗
PubMed1998

5-Substituted pyrimidines with a 1,5-anhydro-2, 3-dideoxy-D-arabino-hexitol moiety at N-1: synthesis, antiviral activity, conformational analysis, and interaction with viral thymidine kinase.

A new series of anhydrohexitol nucleosides are described. These compounds have a pyrimidine base moiety substituted in the 5-position with a chloro (1b), trifluoromethyl (1c), vinyl (1d), 2-thienyl (1e), ethynyl (1f) or propynyl (1g) substituent. The vinyl, propynyl, and, in particular, the 5-trifluoromethyl analogue showed potent activity against herpes simplex virus (HSV), 1c with a selectivity index of >16000 against HSV-1 and >1000 against HSV-2. Conformational analysis of anhydrohexitol nucleosides using computational methods indicates that these nucleosides occur in an equilibrium between the C1 and 1C form with a DeltaE of 5.9 kJ/mol. When the anhydrohexitol nucleoside is cocrystallized with the HSV-1 thymidine kinase it adopts a 1C conformation, which is opposite to the conformation found for the small molecule alone. The enzyme, apparently, induces a conformational change, and conformational flexibility of an anhydrohexitol nucleoside may be advantageous for recognition by viral enzymes.

Antiviral Agents↗
PubMed1997

N6-cyclopentyl-3'-substituted-xylofuranosyladenosines: a new class of non-xanthine adenosine A1 receptor antagonists.

The present study explores the C-3' site of the 3-deoxy-3-xylofuranosyl ring of nucleoside analogues with an adenine or N6-cyclopentyladenine (CPA) base moiety and evaluates the effect on adenosine receptor affinity. Two series of sugar-modified adenosines, i.e., 3'-amido-3'-deoxyadenosines and 3'-amidated 3'-deoxyxylofuranosyladenines, were synthesized and tested for their affinity at A1 and A2a receptors in rat brain cortex and rat striatum, respectively. The modest affinity found in the "xylo series" prompted us to synthesize the corresponding N6-cyclopentyl derivatives, which proved to be well accommodated by the A1 receptors with potencies in the lower nanomolar range. This represents a new perspective in the purinergic field. The absence of a GTP-induced shift, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP indicates an antagonistic behavior of this new class of CPA analogues.

Adenosine↗
PubMed1997

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.

Chromosome Inversion↗
PubMed1997

Screening a random pentapeptide library, composed of 14 D-amino acids, against the COOH-terminal sequence of fructose-1,6-bisphosphate aldolase from Trypanosoma brucei.

A random pentapeptide library composed of 14 D-amino acids, including two unusual amino acids, thus representing 537,824 different peptide sequences anchored on polystyrene beads was created with each bead bearing a single pentapeptide sequence. This library was used for affinity screening against the fructose-1, 6-bisphosphate aldolase of Trypanosoma brucei labeled with biotin as well as versus the COOH-terminal labeled with fluorescein isothiocyanate. The thus selected peptide beads were identified and the appropriate sequences synthesized as peptide amides and evaluated for enzyme activity inhibition. Screening against the whole enzyme did not result in selection of an enzyme inhibitor. However, we demonstrate here that screening against a part of the enzyme involved in the catalytic activity may lead to the discovery of an enzyme inhibitor as well as an enzyme activator. Two low affinity inhibitors, RRVKF-NH2 and KThiKAR-NH2, with an IC50 of approximately 1 mM and approximately 0.2 mM, respectively, were identified. Two other pentapeptides with the sequence SWChaKK-NH2 and SKChaKM-NH2 are able to activate the enzyme fructose-1, 6-bisphosphate aldolase. Thus, successful screening of solid phase libraries can be accomplished using selected sequences of the target enzyme.

Amino Acid Sequence↗
PubMed1997

alpha-Amino acids derived from ornithine as building blocks for peptide synthesis.

Recently great interest has arisen in the synthesis of combinatorial libraries, and this technology provides a significant partner to contemporary strategies in rational design and lead discovery. By simple combination of a given set of building blocks, high numbers of different molecules are produced simultaneously, increasing the possibility of discovery of a lead compound in a limited time. One direction of research in this field focuses on the synthesis of libraries composed of modified amino acids. Here, the synthesis and characteristics of some building blocks derived from ornithine are described. The synthesis is based on the acylation/sulfonation of the copper complex of ornithine by aroyl and arylsulfonyl chlorides exemplified by 2-thiophenecarbonyl chloride, p-toluenesulfonyl chloride and 8-quinolinesulfonyl chloride. To evaluate the potential use of these modified alpha-amino acids as component in an oligopeptide library, all three derivatives were incorporated in a hexapeptide with a random sequence using a standard coupling procedure (DIC/HOBt/DIEA). Depending upon the acidity of the amido hydrogen on the delta-nitrogen, competition between intramolecular cyclization and peptide bond formation was observed. The higher the acidity, the more pronounced is this side reaction. Coupling conditions for peptide formation were optimized so that the newly described amino acid based building blocks are suitable for incorporation into libraries consisting of unnatural amino acids. The outlined procedures open up a broad avenue of possibilities for creation of diversity into peptidic libraries.

Amino Acids↗
PubMed1996

Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry.

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3'- and 5'- terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates. Algorithms for sequence derivation are embodied in a computer program that requires < 2s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direct sequencing of components of mixtures is shown using an unresolved fraction of unknown oligonucleotides from ribosomal RNA.

Algorithms↗
PubMed1995

An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.

Base Sequence↗
PubMed1995

Synthesis and structure-activity relationships of analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a selective inhibitor of trypanosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase.

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.

Animals↗
PubMed1995

Synthesis, biological evaluation, and structure analysis of a series of new 1,5-anhydrohexitol nucleosides.

In view of the selective anti-HSV activity of 1,5-anhydro-2,3-dideoxy-2- (5-iodouracil-1-yl)-D-arabino-hexitol, a series of novel 1,5-anhydrohexitol nucleosides were synthesized and evaluated for their inhibitory activity against several viruses. The 5-iodouracil 3 and the 5-ethyluracil 4 derivatives are highly selective TK-dependent inhibitors of HSV-1 and HSV-2. Broad anti-herpes virus activity was noticed for 5-fluorocytosine 6 and 2,6-diaminopurine 10 analogues. From a transport study of 3, using the thymidine influx competition method, one can conclude that intracellular uptake of this compound most probably occurs by passive diffusion. X-ray analysis of compounds 3 and 9 showed that the heterocyclic base of 1,5-anhydrohexitol pyrimidine and purine is placed in the axial position and that the sugar ring adopts a slightly distorted chair conformation.

Animals↗
PubMed1995

Incorporation of 2'-amido-nucleosides in oligodeoxynucleotides and oligoribonucleotides as a model for 2'-linked conjugates.

The functionalisation of oligodeoxynucleotides and oligoribonucleotides by incorporation of 2'-amido-2'-deoxyribonucleosides, possibly containing a reporter group via the 2'-amido bond, was examined. Therefore 2'-acetamido-ribonucleosides containing a small methyl group at the 2'-amido bond were synthesized as model compounds. In order to evaluate the influence of this 2'-modification on the hybridization capacities, 2'-acetamido-2'-deoxyuridine was incorporated in both RNA and DNA strands. The suitability of phosphoramidite chemistry for the introduction of this modified nucleoside was proven using laser desorption mass spectrometry of the final oligonucleotide. The presence of the 2'-modification destabilised both RNA-RNA, DNA-DNA and mixed duplexes. Therefore, it can be concluded that the 2'-acetamido group is not a good linker for attachment of reporter groups to oligonucleotides.

Base Sequence↗
PubMed1995

Identification of a peptide inhibitor against glycosomal phosphoglycerate kinase of Trypanosoma brucei by a synthetic peptide library approach.

A synthetic peptide library, composed of 2.5 million L-amino acid pentapeptides anchored on polystyrene beads was prepared with each bead bearing a single pentapeptide sequence. This library was screened for interaction with glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei labelled with fluorescein or with biotin. Affinity beads that bound the enzyme were selected with a pipette or with streptavidin coated magnetic beads. The beads that bound to the enzyme were individually subjected to Edman microsequence analysis to determine the sequence of the corresponding peptide ligands. The corresponding peptide-sequences were synthesised as free peptide acids and evaluated for enzyme activity inhibition. The pentapeptide NWMMF was able to selectively inhibit gPGK with an IC50 of approximately 80 microM.

Affinity Labels↗
PubMed1993

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.

Base Composition↗
PubMedDate not supplied

New techniques for the rapid characterization of oligonucleotides by mass spectrometry.

Recent advances in combined HPLC/electrospray ionization-mass spectrometry provide effective new capabilities for the rapid characterization of oligonucleotides. Accurate mass measurements with errors < 0.3 Da, and determination of base and sugar modification and of nearest neighbor identities, can be routinely carried out on 10-100 component mixtures of RNA or DNA. These procedures are widely applicable in structural and analytical studies involving mixtures of oligonucleotides.

Chromatography, High Pressure Liquid↗