The Interaction of Trihalogenoacetic Anhydrides and Trihalogenoacetyl Chlorides with Thymidine 5"-Phosphate as an Approach to New Activating Agents in the Phosphorylation Reactions for Nucleotides

The interaction of thymidine 5"-phosphate with trichloroacetic anhydride, trichloroacetyl chloride, and tribromoacetyl bromide was studied in dimethylformamide and acetonitrile in the presence of tertiary amines. The first two reactions gave the mixed anhydride of trichloroacetic and thymidylic acids (acyl phosphate) as the major product and P ¹,P ²-dithymidine 5"-pyrophosphate as the byproduct. The third reaction proceeded by a more complicated mechanism and mainly led to substituted polyphosphates. The subsequent treatment of the reaction mixtures with morpholine resulted in thymidine 5"-phosphoromorpholidate in a high yield. The phosphorylating activities of the trichloroacetyl and tribromoacetyl phosphates were 77 and 89%, respectively.     

The regulation of thymidine secretion by macrophages.

The secretion of thymidine by mononuclear phagocytes was correlated with the activity of the enzyme thymidine kinase (TK). Macrophages cultured in regular tissue culture medium released thymidine and did not express TK. However, when macrophages were incubated with medium conditioned by L cells, they expressed TK, incorporated 3H thymidine into trichloroacetic acid precipitable material, and ceased to secrete the nucleotide. Furthermore, replicating P388/D1 cells were induced to secrete thymidine by inhibiting TK with d-glucosamine. These results have demonstrated an inverse relationship between thymidine secretion and the expression of TK. They suggest that thymidine secretion by macrophages may be attributed to their lack of TK activity.     

Synthesis and reactions of a nucleoside derivative of phosphoric sulfonic anhydride. Studies related to the mechanisms of coupling reactions in the chemical synthesis of oligodeoxyribonucleotides by phosphotriester procedures

The synthesis of a model compound, diphenylphosphoric toluene-p-sulfonic anhydride, an arylsubstituted phosphoric sulfonic mixed anhydride, is described. Using the same procedure a thymidyl substituted derivative was prepared. The phosphoric sulfonic anhydride is the presumed intermediate in oligonucleotide coupling reactions involving phosphodiester activation by arenesulfonyl derivatives. This mixed anhydride reacts with a variety of nucleophiles. It can be converted to phophotriester derivatives in the presence of simple alcohols. Phosphotriester formation using the 5'-hydroxyl of a thymidine derivative requires additionally a catalyst such as N-methylimidazole. The reactive intermediate produced upon the addition of N-methylimidazole to the phosphoric sulfonic anhydride has been observed spectroscopically using 31P-NMR.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Preferential uptake of thymidine by thymineless enterobacteria: its significance in DNA labelling.

Minimum satisfactory concentrations of thymine and thymidine were determined for the growth of a high thymine-requirng (thy) mutant to Escherichia coli strain J5-3. Cultures were then grown in the presence of these concentrations of non-radioactive ('cold') pyrimidine together with 5 microCi/ml [methyl-3H)thymine, or [methyl-3H)thymidine (specific activities 5 Ci/m mole), and the uptake of radioactivity into ice cold trichloroacetic acid insoluble material determined. By far the most efficient labelling system was obtained if the label was supplied as radioactive thymidine and growth requirements satisfied by thymine alone. The addition of deoxyadenosine to the labelled thymidine/unlabelled thymine system dramatically reduced uptake of label. The addition of radioactive thymine with either thymine or thymidine to ensure satisfactory growth gave poor labelling. Using the [methyl-3H] thymidine/thymine system it was possible to increase the concentration of thymine from 8 to 64 microgram/ml with only a 25% reduction in label uptake after a 2 h period. The same system was also shown to be most efficient for labelling a thy derivative of another K12 strain, a thymine low-requiring (tir) K12 strain, a thy mutant of Klebsiella aerogenes 418 and a tir derivative of Salmonella typhimurium LT2.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Polyamine-induced DNA Synthesis and Mitosis in Oat Leaf Protoplasts

Freshly isolated protoplasts from leaves of oat seedlings (var. Victory) which do not divide when cultured on a wide range of media are capable of incorporating tritiated leucine, uridine, and thymidine into trichloroacetic acid-insoluble macromolecules. Over 70% of the leucine and uridine incorporated over an 18-hour period are found in protein and RNA, respectively, as shown by hydrolysis of the macromolecular products with a specific protease or RNase. In contrast, little or none of the tritiated thymidine is incorporated into macromolecules hydrolyzable by DNase over an 18- to 96-hour period. Incorporation of thymidine into trichloroacetic acid-insoluble material declines sharply with increasing time of culture after 18 hours. However, addition of diamines or polyamines to the medium not only prevents the decline, but actually increases net thymidine incorporation, including a fraction going into DNA. A significant increase in mitoses and binucleate protoplasts is also observed in 72- to 168-hour cultures.     

Improved catalytic and stereoselective glycosylation with glycosyl N-trichloroacetylcarbamate: application to various 1-hydroxy sugars

Efficient catalytic and stereoselective glycosylation was achieved by activating a glycosyl N-trichloroacetylcarbamate with a catalytic amount of Lewis acid in the presence of a glycosyl acceptor and 5 ? molecular sieves. Catalytic one-pot dehydrative glycosylation of a 1-hydroxy carbohydrate was achieved stereoselectively by reaction with trichloroacetyl isocyanate, followed by activation with a catalytic amount of activators.     

Reaction of uracil with hypochlorous acid.

The end products of the reaction of uracil with at least a 10-fold excess of aqueous hypochlorous acid at pH 7–8 were found to be trichloroacetic acid, carbon dioxide and nitrogen trichloride. Little formation of trichloroacetic acid was observed after 24 hours when the ratio of hypochlorous acid to uracil was less than 4:1. An intermediate in the reaction was found to be 5-chlorouracil. This was also degraded by hypochlorous acid to trichloroacetic acid.     

Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Two Phases in Adventitious Root Formation in Phaseolus mungo Hypocotyl Cuttings, a Phase Sensitive to an Inhibitor of RNA or Protein Synthesis and a Phase Sensitive to an Inhibitor of DNA Synthesis

The development of adventitious roots in Phaseolus mungo cuttingswas inhibited by 2-thiouracil, cycloheximide, and 5-bromodeoxyuridine.The stage of rooting blocked by 2-thiouracil and cycloheximidewas different from that blocked by 5-bromodeoxyuridine. Somecell division in the basal rooting region occurred with 5-bromodeoxyuridine,but not with 2-thiouracil and cycloheximide. Radioactivity from labelled 2-thiouracil appeared in RNA fractionsbut the amount was reduced by simultaneously applied uracil.5-Bromodeoxyuridine inhibited incorporation of thymidine intoDNA fractions. 2-Thiouracil and 5-bromodeoxyuridine act as antimetabolitesof uracil and thymidine, respectively. Cycloheximide, an inhibitorof protein synthesis, prevented the incorporation of radioactivityfrom labelled leucine into the trichloroacetic acid-insolublefraction. RNA synthesis inhibitors (2-thiouracil and actinomycin D) andprotein synthesis inhibitors (cycloheximide and blasticidinS) increased roots effectively when dosed at the beginning ofincubation. Inhibitors of DNA synthesis (5-bromodeoxyuridineand mitomycin C) were effective when applied after several hours'pre-incubation in water. It is suggested that there are at leasttwo phases in adventitious root formation, a phase sensitiveto an inhibitor of RNA or protein synthesis and a phase sensitiveto an inhibitor of DNA synthesis.     

A method of the rapid preparation of adenosine 5'-gamma-[32P] triphosphate by chemical synthesis.

A new chemical method for the synthesis of adenosine 5'-gamma-[32P] triphosphate has been developed based on the reaction of adenosine 5'-diphosphate with ethyl chloroformate. The resulting active mixed anhydride was able to react with [32P]-triethylammonium orthophosphate to give gamma-[32P]ATP.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Assessment of [3H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25°C, but hydrolysis at 120°C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Phospho-carboxylic anhydride of a homologated nucleoside leads to primer degradation in the presence of a polymerase

Starting from thymidine, through a series of key synthetic transformations (e.g., Wittig reaction, hydroboration, Mitsunobu reaction and TEMPO oxidation) a nucleoside homologue bearing a phospho-carboxylic anhydride group at 6′ position was synthesized. The potential of polymerases to catalyze amide bond formation was investigated by using a modified primer with an amino group at 3′ position and the synthesized phosphoanhydro compound as substrate. Unfortunately, we did not observe the desired product either by gel electrophoresis or mass spectrometry. In contrast, the instability of the phosphoanhydro compound could lead to pyrophosphate formation and thus, to pyrophosphorolysis of the primer rather than to primer extension.     

Isolation of two biologically active peptides, erythrotropin I and erythrotropin II from fetal calf intestine

A bioassay based on the measurement of thymidine incorporation into trichloroacetic acid-insoluble materials in erythroid cell suspensions from fetal calf liver was used as the assay for purification of two small peptides (erythrotropins I and II) from fetal calf intestine. The peptides were purified using reversed-phase and gel permeation high performance liquid chromatography (HPLC). The two peptides have very similar amino acid compositions and a molecular weight of about 3500 daltons. Erythrotropin II stimulated thymidine incorporation and potentiated the action of erythropoietin in cultures of erythroid cells from fetal rat liver.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Acetic anhydride: an intermediate analogue in the acyl-exchange reaction of citramalate lyase.

1. Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzyme-anhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride. 2. A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S-citramalate appears to be non-competitive with iodoacetate. 3. The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lyase. 4. The formation of 6-citryl thiolester from the 1-thiolester via the cyclic citric anhydride provides a chemicla model for enzymic acyl exchange. 5. The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters.     

Iodine Monochloride Facilitated Deglycosylation,Anomerization, and Isomerization of 3-Substituted Thymidine Analogues

The reaction of thymidine, 3-mono-, and 3,3′,5′-trialkylsubstitued thymidine analogues with iodine monochloride (ICl) was investigated. Treatment with ICl resulted in rapid deglycosylation, anomerization, and isomerization of thymidine and 3-substituted thymidine analogues under various reaction conditions leading to the formation of the nucleobases as the major products accompanied by minor formation of α-furanosidic-, α-pyranosidic-, and β-pyranosidic nucleosides. On the other hand, 3,3′,5′-trisubstitued thymidine analogues were only deglycosylated and anomerized. These results are similar to those observed for the acidic hydrolysis of the glycoside bond in nucleosides, but were presumably caused by the Lewis acid character of an iodine electrophile.     

Determining [H]Thymidine Incorporation into Bacterioplankton DNA: Improvement of the Method by DNase Treatment

Determination of [H] thymidine incorporation into bacterial DNA versus other macromolecules is usually achieved by NaOH and hot trichloroacetic acid hydrolysis. This procedure was found not to be specific enough. An alternative method founded on DNase treatment is proposed. Under the new method, the fraction of thymidine incorporated into DNA ranged from 10 to 83%.