5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.     

Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

Metabolism of Pyrimidine Nucleotides in a Microorganism: III. Enzymatic Production of Ribose-5-Phosphate from Uridine-5′-Monophosphate by Pseudomonas oleovorans

A study was made to develop a new method for the production of ribose-5-phosphate (R-5-P) from uridine-5'-monophosphate (UMP) by the action of nucleotide-N-ribosidase of Pseudomonas oleovorans, and a suitable medium for the formation of nucleotide-N-ribosidase was established. For the enzymatic conversion of UMP to R-5-P, a cell suspension was employed as the enzyme source. Although degradation of R-5-P, the desired product, occurred during the course of the enzyme reaction, it was prevented by the addition of an appropriate amount of zinc ion and resulted in a stoichiometric conversion of UMP to R-5-P and uracil. Accumulated R-5-P was readily isolated by ion-exchange chromatography of the bacteria-free reaction mixture. Yield of isolated R-5-P was about 60% of the theoretical recovery.     

Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.     

Uridine-5'-phosphate synthase: evidence for substrate cycling involving this bifunctional protein

Uridine 5'-phosphate (UMP) synthase contains two sequential catalytic activities for the synthesis of orotidine 5'-phosphate (OMP) from orotate (EC 2.4.2.10, orotate phosphoribosyltransferase) and the decarboxylation of OMP to form UMP (EC 4.1.1.23, OMP decarboxylase). Previous kinetic studies had indicated that partial channeling of OMP might occur [T.W. Traut and M.E. Jones (1977) J. Biol. Chem. 252, 8374-8381]; in the presence of a nucleotidase, there was no measurable formation of orotidine from OMP under conditions where OMP was maintained at a steady-state concentration [T.W. Traut (1980) Arch. Biochem. Biophys. 200, 590-594]. Recently claims were made that (i) the steady-state activities of UMP synthase could be modeled by Michaelis-Menten kinetics, and (ii) the nucleotidase activity in Ehrlich ascites cells was insufficient to degrade any significant amount of OMP [R.W. McClard and K.M. Shokat (1987) Biochemistry 26, 3378-3384]. The present studies show that UMP synthase has cooperative kinetics toward OMP, and that a substrate cycle involving orotate phosphoribosyltransferase, cytoplasmic nucleotidase, and uridine phosphorylase maintains the cyclic interconversion: orotate----OMP----orotidine----orotate, etc. It is therefore the complex steady-state kinetics of UMP synthase in the presence of OMP, and the existence of a substrate cycle that account for the results which were interpreted as channeling in the earlier studies.     

Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate

A procedure was devised to detect and assay uridine 5′-pyrophosphate (UDP)-glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP-glucuronic acid and ³²P-pyrophosphate, and the ³²P-uridine 5′-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine ³²P-adenosine 5′-triphosphate produced by adenosine 5′-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5′-triphosphate.     

Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents

A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against a panel of ten RNA and DNA viruses.Fifty-two of them displayed different levels of activity against one or more viruses, among which CVB-5, RSV, BVDV and Sb-1 were the most frequently affected. In particular, fourteen compounds exhibited an EC₅₀ in the range 9–17 μM (SI from 6 to >11) versus CVB-5, and seven compounds showed an EC₅₀ in the range 5–15 μM (SI from 6.7 to ⩾20) against RSV, thus resulting comparable to or more potent than the respective reference drugs (NM108 and ribavirin). Most of these compounds derive from 2-benzylbenzimidazole, but also other molecular scaffolds [as 1-phenylbenzimidazole (2), 2-trifluoromethylbenzimidazole (69), dihydropyrido[3′,2′:4,5]imidazo[1,2-a][1,4]benzodiazepin-5-one (3), dibenzo[c,e]benzimidazo[1,2-a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazole (81, 82 and 86)] are related to interesting levels of activity against these or other viruses (BVDV, Sb-1). Thus, these scaffolds (some of which, so far unexplored), represent valid starting points to develop more efficient agents against pathologies caused by CVB-5, RSV, BVDV and Sb-1 viruses.     

Synthesis and biological studies of 5-aminolevulinic acid-containing dendrimers for photodynamic therapy.

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)-containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3'-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.     

Amino acid sequence of toxin III from Anemonia sulcata.

Toxin III, the smallest toxin component of the poison of the sea anemone Anemonia sulcata, is a polypeptide with 27 amino acids. Its structure is stabilized by three disulfide bridges. The amino acid sequence was determined by solid-phase Edman degradation of the aminoethylated derivative. The peptide was coupled to the carrier, porous glass, by thiourea bridges between the alpha-amino group of arginine-1 and the epsilon-amino group of lysine-26 and the isothiocyanate groups of the carrier. Another fraction of the polypeptide was bound by an acid-amide condensation of the C-terminal valine-27 with the aminopropyl group of the carrier. The sequence of toxin III has no regions homologous to the 47-residue toxin II. Comparison with the known partial sequence of toxin I, which contains 46 amino acids (Wunderer, G. & Eulitz, M., in preparation) also fails to reveal homologies.     

Amino acid and peptide derivatives of dimethyl 5-aminoisophthalate as fluorogenic substrates for proteinases

A number of amino acid and peptide derivatives of the fluorophore, dimethyl 5-aminoisophthalate have been synthesized, characterized and tested as substrates for the plant cysteine proteinases papain, ficin and bromelain. In every case, replacement of alanine by citrulline, in the position adjacent to the dimethyl 5-aminoisophthalate resulted in a higher rate of hydrolysis. The partly deprotected dipeptide derivative dimethyl phenylalanylcitrulline-5-aminoisophthalate was hydrolysed most rapidly of all the compounds tested, and on this basis may provide a useful substrate for the detection and quantitative assay of these enzymes.     

Topography and Function of Golgi Uridine-5[prime]-Diphosphatase from Pea Stems

Golgi UDPase is an enzyme that has been shown to function in polysaccharide biosynthesis, but its role in this process is not yet clear. In this study we identified Golgi UDPase activity in pea (Pisum sativum) stems and differentiated it from another UDPase activity. We demonstrated that Golgi UDPase is an integral membrane protein, based on specific partitioning of this activity into Triton X-114. Analysis of its topology using sealed, right-side-out Golgi vesicles and treatment with proteinase K suggested that its active site faces the Golgi lumen. Studies aimed at understanding the function of Golgi UDPase by incubating Golgi vesicles with [beta]-32P]UDP-glucose (Glc) to generate [beta]-32P]UDP upon Glc transfer in situ showed that 32Pi, but not [beta]-32P]UDP, was formed, suggesting that UDPase quickly hydrolyzed the UDP formed during Glc polymerization. We found that the Golgi UDPase was highly active in the elongating region of the third internode, whereas no activity was detected in the first and second internodes of etiolated pea seedlings. These results suggest that UDPase removes the UDP formed during Glc polymerization and could be important in the mechanism of polysaccharide biosynthesis.     

Amino acid analysis utilizing phenylisothiocyanate derivatives

Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.     

Structure determination of two new amino acid-containing derivatives of adenosine from tRNA of thermophilic bacteria and archaea.

Two new nucleosides have been identified in unfractionated transfer RNA of two thermophilic bacteria, Thermodesulfobacterium commune, and Thermotoga maritima, six hyperthermophilic archaea, including Pyrobaculum islandicum, Pyrococcus furiosus and Thermococcus sp. and two mesophilic archaea, Methanococcus vannielii and Methanolobus tindarius. Structures were determined primarily by mass spectrometry, as 3-hydroxy-N-[[(9-beta-D-ribofuranosyl-9H-purin-6- yl)amino]carbonyl]norvaline, (hn6A), structure 1, and 3-hydroxy-N-[[(9-beta-D-ribofuranosyl-9H-2-methylthiopurin-6- yl)amino]carbonyl]norvaline (ms2hn6A), 2. The amino acid side chain was characterized as 3-hydroxynorvaline (3) by gas chromatography-mass spectrometry of the trimethylsilyl derivative after cleavage from 1 and 2 by alkaline hydrolysis. Evidence for the amino acid-purine carbamoyl linkage was obtained from the collision-induced dissociation mass spectrum of trimethylsilylated 1, and the total structure was confirmed by chemical synthesis of 1.     

Autoradiographic Studies of Uridine-5-H3 Uptake in Entamoeba histolytica in the CLG Medium1

SYNOPSIS. Using uridine-5-H³, “long-term” labeling experiments over a 72 hr growth cycle were done with E. histolytica strain K9 grown in CLG medium with penicillin-inhibited Bacteroides. Autoradiographic analysis revealed that tritium occurs primarily in cytoplasm and rarely the nucleus of amebae. The most extensive cytoplasmic activity was observed during the initial 0–24 hr growth period of amebae as compared to later labeling periods. RNase or RNase followed by DNase extracted a large amount but not all label from amebae. These nucleases were least effective during the initial 24 hr period of growth. Thus it appears that tritium from uridine-5-H³ is not highly specific for RNA in amebae. However, the possibility that such label is associated with RNase-resistant RNA cannot be ruled out. More recent cytochemical studies do indicate the presence of RNase-resistant RNA in the cytoplasm of amebae. The activity found in penicillin-inhibited Bacteroides after uridine-5-H³ labeling and their reaction to the various digestive procedures was similar to amebae at corresponding labeling periods. Therefore at least some of the RNase-resistant material present in the cytoplasm of amebae may be derived from the ingested bacteria; this has been further found by appropriate experiments in which amebae were fed prelabeled bacteria. Nuclear activity when observed (always after 24 hrs growth) was associated either with the periphery of the nucleus and/or the endosome. It was not seen in the nuclear stroma. Some of this activity is RNase-resistant, perhaps representing double or multi-stranded RNA. It therefore appears that RNA is not distributed in the nuclear stroma in “long-term” labeling experiments.