Biomedical subjects
Publications and source records attributed to Jef Rozenski.
A novel class of inhibitors of pestiviruses (5-substituted 2-phenyl-5H-imidazo[4,5-c]pyridines) is described. Modification of the substituent in position 5 resulted in analogues with high activity (EC(50)<100nM) and selectivity (SI>1000) against the pestivirus BVDV (bovine viral diarrhea virus).
During synthesis of thiolated deoxyoligonucleotides, side products can be formed. When used in the fabrication of microchips, the oligonucleotides have to be of high purity. We demonstrated the presence of impurities, which were not failure sequences from the synthesis. These products were identified and characterized using high-performance liquid chromatography (HPLC) and electrospray MS(/MS). The presence of the free thiol group was assessed by acrylamide derivatization. After reaction with acrylamide the correct compounds showed a 71 u mass shift and the major fragment ions could be assigned as 5' a-base and 3' w ions, similar as for unmodified DNA. The side products were unaffected by acrylamide, suggesting that the thiol group was modified. The oligonucleotide containing a species with a mass of 32 u higher was identified as 5' sulfinic acid containing molecule and was found as 45% of the total amount of a DNA 25 mer. Other components appeared to be dithio-linked oxidation products, present about 1 to 5% in a 10 mer and 15 mer deoxyoligonucleotide. The analyses were useful for optimizing the synthesis protocols.
In this study we report the design, synthesis, and activity against bovine viral diarrhea virus (BVDV) of a novel series of acridone derivatives. BVDV is responsible for major losses in cattle. The virus is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. Some of the synthesized acridones elicited selective anti-BVDV activity with EC(50) values ranging from 0.4 to 4 microg/mL and were not cytotoxic at concentrations that were 25- to 200-fold higher (CC(50) >100 microg/mL). It was proven that the most potent acridone derivative 10 was able to not only protect cells from virus-induced cytopathic effect but also reduce the production of infectious virus and extracellular viral RNA. Furthermore, compound 10, as well as a number of other analogues, inhibited HCV replication to some extent. However, there was no direct correlation between anti-BVDV and anti-HCV activity. Thus, the acridone scaffold, when appropriately functionalized, can yield compounds with selective activity against pestiviruses and related viruses such as the HCV.
A new series of hybrid PDMP analogues, based both on PDMP and styryl analogues of natural ceramide, has been synthesized from D-serine. The synthetic route was developed such that future introduction of different aryl groups is straightforward. Biological evaluation, both in vitro on rat liver Golgi fractions as well as in HEK-293 and COS-7 cells, revealed two lead compounds with comparable inhibitory potency as PDMP, which could be elaborated to more potent inhibitors.
We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.
Hypericin, a naturally occurring hydroxylated phenanthroperylene dione, is used as a powerful photosensitizer for photodynamic therapy as well as a diagnostic tool for the fluorescence detection of flat neoplastic lesions in the bladder of patients. Both applications are based on the tumouritropic characteristics of the compound. To get more insight into some of the physicochemical properties of hypericin affecting its tumouritropic characteristics, we set out to synthesize a series of more lipophilic hypericins. For this purpose, a synthetic pathway to hypericin acid amides with hydrocarbon chains of different lengths stably attached by an amide bond at position C10 was explored. Hypericin acid proved inert in amide forming reactions, whereas the precursor protohypericin acid showed higher reactivity and resulted in the desired amide derivatives, which afterwards can be easily converted into their phenanthroperylene dione form. Hexyl-, octyl-, decyl- and dodecylamides of hypericin acid were successfully synthesized in this way. In vitro cellular uptake and photo-induced antiproliferative effects of the compounds were evaluated, using the human moderately differentiated non-invasive papillary transitional carcinoma RT-112 cell line. Whereas the more lipophilic amides were taken up limitedly, the hexylamide accumulated approx. as well as hypericin itself. From the antiproliferative data it can further be concluded that not only the cellular uptake, but also the light-induced activity, is affected by the introduced structural changes.
A method is presented for improved detection of pseudouridine in nucleoside mixtures based on the specific derivatization with methyl vinyl sulfone followed by analysis by capillary HPLC-mass spectrometry. Reaction conditions were optimized in order to obtain the best yield and specificity. The method was successfully applied to different nucleoside mixtures.
Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.
The Small Subunit rRNA Modification Database provides a listing of reported post-transcriptionally modified nucleosides and sequence sites in small subunit rRNAs from bacteria, archaea and eukarya. Data are compiled from reports of full or partial rRNA sequences, including RNase T1 oligonucleotide catalogs reported in earlier literature in studies of phylogenetic relatedness. Options for data presentation include full sequence maps, some of which have been assembled by database curators with the aid of contemporary gene sequence data, and tabular forms organized by source organism or chemical identity of the modification. A total of 32 rRNA sequence alignments are provided, annotated with sites of modification and chemical identities of modifications if known, with provision for scrolling full sequences or user-dictated subsequences for comparative viewing for organisms of interest. The database can be accessed through the World Wide Web at http://medlib.med.utah.edu/SSUmods.
Oligonucleotides that contain up to three aminopropyl nucleoside analogues have been synthesized. Dimers of aminopropyl adenine and thymidine were prepared and used as building blocks by applying phosphoramidite chemistry. Both R and S isomers of the aminopropyl nucleosides were used. This incorporation led to a reduction of thermal stability of double-stranded DNA. Furthermore, the (R)-adenine analogue, which yielded (S)-APNA, can be considered as a candidate for universal base pairing.
An enzymatic transesterification reaction afforded large scale resolution of the cyclohexenol precursor needed for preparation of both series of CeNA building blocks. CeNA oligos of "D-like" chirality display a strong and selective interaction with RNA, while preserving RNase H activity, and therefore have potential as antisense constructs. CeNAs of opposite chirality form a self-pairing system on their own.
Thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for selectively blocking bacterial DNA synthesis. Hereby, we report on the discovery of a novel class of bicyclic nucleosides (10 and 11) and one dinucleoside (12), belonging to the most selective inhibitors of TMPKmt discovered so far.
[structure: see text] Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting sequence information at the outside of the helix.
A ribose residue inserted between the 3'-OH of one nucleotide and the 5'-phosphate group of the next nucleotide, functions as a site-specific cleavage site within DNA. This extra ribose does not interrupt helix formation and it protects duplex DNA against cleavage by restriction enzymes. Cleavage can be obtained with periodate and all ribose fragments can be removed with sodium hydroxide. As a result of this, an intact natural oligodeoxynucleotide is obtained after ligation reaction, which means that site-specific cleavage and recovering of intact DNA occurs without loss of genetic information.
Alpha-L-threose nucleic acids (TNA) are potentially natural nucleic acids that could have acted as an evolutionary alternative to RNA. We determined whether DNA or RNA polymerases could recognize phosphorylated threosyl nucleosides. We found that for both the Vent (exo-) DNA polymerase and HIV reverse transcriptase K(m) values were increased and kcat values decreased for the incorporation of tTTP in comparison to their natural counterparts. Our results suggest that TNA may have played a role in the evolution of the DNA-RNA-protein world. Thus, TNA may be a candidate for further studies in evolutionary chemistry and biology.
The synthesis of fully [13C/15N] labeled pyrimidine nucleosides has been achieved from 13C-glucose and labeled nucleobases. The reaction scheme leads directly to the protected nucleosides without the need for the inversion of configuration of C-3 of 13C-glucose. This was achieved by an oxitative ring-opening reaction removing the carbon with the wrong configuration.
From excreta of chickens that had been treated with sodium salicylate, a new compound was detected and identified as a double conjugated ornithine metabolite. The structural assignment of this metabolite was further confirmed by an independent efficient 3-step synthesis from ornithine.
A strategy was developed for the synthesis of 3'-O-beta-D-ribofuranosyl 2'-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5'-O- and 5''-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.