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Methods for direct synthesis of N-(purin-8-yl)arylamines were investigated. N-(Purin-8-yl)arylamines are adducts from reaction of electrophilic metabolites of arylamines with DNA and have not been readily available by direct synthesis. Ability to generate significant quantities of this class of DNA adduct by synthetic means would facilitate basic research in molecular toxicology. Two routes with common thiourea intermediates were developed for this purpose. In the first route, 6-hydroxy-2,4,5-triaminopyrimidine was caused to react in aqueous medium with dithiocarbamate derivatives of o-, m-, or p-toluidine to form corresponding 2,4-diamino-5-(tolylthioureido)aminopyrimidin-6-ones in 60-70% yields. The thiourea intermediates were converted to carbodiimides and isolated in 20-25% yields after treatment with HgO in dimethylformamide. The carbodiimides were cyclized at 125 degrees C in dimethylformamide under N2 for 45 h to yield N-(guanin-8-yl)(o-, m-, or p-)toluidines in 65-75% yields. In the second route, direct reaction of p-tolyl isothiocyanate with 6-hydroxy-2,4,5-triaminopyrimidine or with 4,5,6-triaminopyrimidine in dimethylformamide and triethylamine led to the formation of 2,4-diamino-5-(p-tolylthioureido)aminopyrimidin-6-one and 4,6-diamino-5-(tolylthioureido)aminopyrimidine, respectively, in 77-79% yields. Methylation of the two thiourea derivatives by methyl iodide in dimethylformamide gave the corresponding methylisothiuronium derivatives, which were isolated in 71% and 84% yields (as HI salts), respectively. Conversion of the methylisothiuronium derivatives to N-(guanin-8-yl)-p-toluidine or N-(adenin-8-yl)-p-toluidine was accomplished by heating in dimethylformamide for 5-7 h at 80-90 degrees C, in 70% and 62% yields, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
A new N-protecting group, ethanesulfonylethoxycarbonyl (Esc), was designed to perform peptide synthesis in both aqueous and organic solvents. Esc-amino acids were prepared by the reaction of Esc-Cl and amino acids. Although Esc-Cl was a highly reactive reagent, it was not stable and decomposed during the purification procedure. A more stable reagent, ethanesulfonylethyl-4-nitrophenyl carbonate (Esc-ONp), was designed for preparation of Esc-amino acids. Esc-ONp was a stable reagent and could be purified by silica gel column chromatography or recrystallization. Esc-amino acids were prepared by the reaction of Esc-ONp and amino acids in good yield. To evaluate Esc-amino acids, Leu-enkephalin amide was synthesized using Esc-amino acids by the solid phase method in water. Removal of the Esc group was performed with 0.025 mol/l NaOH in 50% aqueous ethanol. Leu-enkephalin amide was successfully synthesized on a poly(ethylene glycol)-grafted polystyrene resin. Esc-amino acids have moderate solubility in organic solvents (such as dimethylformamide and acetonitrile). Leu-enkephalin amide was synthesized using Esc-amino acids by the solid phase method in dimethylformamide. Removal of the Esc group was performed with 0.05 mol/l tetrabutylammonium fluoride in dimethylformamide. Synthesis of Leu-enkephalin amide using Esc-amino acids in dimethylformamide was also successful. The yields of synthesis of Leu-enkephalin amide in water and dimethylformamide were 71% and 67%, respectively.
The effects of dexamethasone, a synthetic glucocorticoid, and of N,N-dimethylformamide on in vitro growth and differentiation and on proto-oncogene expression of human rhabdomyosarcoma cells were studied. RD/18 clone cells (derived from the embryonal rhabdomyosarcoma cell line RD) treated with 100 nM dexamethasone showed an almost complete block of differentiation: about 5% myosin-positive cells were observed after 2 weeks of culture in dexamethasone-supplemented differentiation medium, compared to 20% of untreated cultures. Dexamethasone also induced a 20-30% growth inhibition and a more flattened morphology. The treatment with N,N-dimethylformamide induced a significantly increased proportion of myosin-positive cells (reaching about 30%) and a 40% growth inhibition. Induction of differentiation inversely correlated with the levels of c-myc proto-oncogene expression: after a 2 week culture dexamethasone-treated cells showed the highest c-myc expression and N,N-dimethylformamide-treated cells the lowest. Culture conditions per se down-modulated c-erbB1 and up-regulated c-jun expression, with no relationship to the differentiation pattern. Other proto-oncogenes were not expressed (c-sis, N-myc, c-mos, c-myb) or were not modulated (c-fos, c-raf). Therefore dexamethasone and N,N-dimethylformamide, both causing a decreased growth rate, showed opposing actions on myogenic differentiation and on c-myc proto-oncogene expression of human rhabdomyosarcoma cells.
Four organic solvents were tested for their ability to interfere with ADP- and 2,4-dinitrophenol-stimulated respiration in tightly-coupled guinea-pig liver mitochondria. All four compounds inhibited mitochondrial respiration, but dimethylformamide and ethanol were more potent than dimethylsulphoxide and propyleneglycol. The latter compound was the only one that failed to produce a significant inhibition of the reactions. Using dimethylformamide, it was shown that inhibition of the reactions was independent of the substrates employed. A detailed analysis was made of the effect of a range of concentrations of dimethylformamide on the uncoupling activity of a range of concentrations of 2,4-dinitrophenol. It was clearly demonstrated that the inclusion of dimethylformamide in the reaction medium would result in an underestimate of the activity of dinitrophenol. Recommendations are made concerning the precautions that should be taken when analysing the effects of water-insoluble drugs on oxidative phosphorylation.
In 2,4-dihydroxybenzaldehyde 2,4-dinitrophenylhydrazone N,N-dimethylformamide solvate [or 4-[(2,4-dinitrophenyl)hydrazonomethyl]benzene-1,3-diol N,N-dimethylformamide solvate], C(13)H(10)N(4)O(6).C(3)H(7)NO, (X), 2,4-dihydroxyacetophenone 2,4-dinitrophenylhydrazone N,N-dimethylformamide solvate (or 4-[1-[(2,4-dinitrophenyl)hydrazono]ethyl]benzene-1,3-diol N,N-dimethylformamide solvate), C(14)H(12)N(4)O(6).C(3)H(7)NO, (XI), and 2,4-dihydroxybenzophenone 2,4-dinitrophenylhydrazone N,N-dimethylacetamide solvate (or 4-[[(2,4-dinitrophenyl)hydrazono]phenylmethyl]benzene-1,3-diol N,N-dimethylacetamide solvate), C(19)H(14)N(4)O(6).C(4)H(9)NO, (XII), the molecules all lack a center of symmetry, crystallize in centrosymmetric space groups and have been observed to exhibit non-linear optical activity. In each case, the hydrazone skeleton is fairly planar, facilitated by the presence of two intramolecular hydrogen bonds and some partial N-N double-bond character. Each molecule is hydrogen bonded to one solvent molecule.
Three organic modifiers, methanol, acetonitrile, and dimethylformamide, were used as organic modifiers to improve the resolution of hydrophobic compounds in micellar electrokinetic chromatography. The capacity factor decreased with increasing concentration of organic modifier. Acetonitrile and dimethylformamide were more effective modifiers to decrease the capacity factor than methanol. Maximum resolution was obtained when the capacity factor was reduced to about 2. Methanol enhanced the resolution for less hydrophobic compounds, while acetonitrile and dimethylformamide were better modifiers to improve the resolution for strongly hydrophobic compounds.
Some methods for analysing N,N-dimethylformamide and its metabolites [hydroxymethyl-N-methylformamide, hydroxymethylformamide and N-acetyl-S-(N-methylcarbamoyl)cysteine] in the urine of exposed workers are described. Unchanged dimethylformamide was measured after pretreatment of urine (2 ml) with silica gel cartridges and elution with methanol. The gas chromatographic analysis using a nitrogen phosphor detector made it possible to detect N,N-dimethylformamide in urine even when workers were exposed to low concentrations of the solvent (about 1 mg/m3). N-Hydroxymethyl-N-methylformamide and N-hydroxymethylformamide were analysed as N-methylformamide and formamide respectively after direct injection of urine into the gas chromatograph. The injection port temperature played an important role in the gas chromatographic determination of these products. Reliable results were obtained when direct or split injections were performed at 250 degrees C. The splitless injection gave the same reliable results at 150 degrees C. In urine samples from occupationally non-exposed persons, N-methylformamide could not be detected. In contrast, formamide (or its precursor, hydroxymethylformamide) was present in every urine sample. Our results in respect of 19 urine samples analysed with the injection port of the gas chromatograph at 250 degrees C gave a mean of 8.6 mg/l of formamide. N-Acetyl-S-(N-methylcarbamoyl)cysteine was determined using a modified method for analysing organic acid in urine samples. The metabolite was extracted with ethyl ether in an acid environment, treated with a silylating reagent and measured by gas chromatography/mass spectrometry.
The N,N-dimethylformamide-hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49-fold purification, a 24% yield and a final specific activity of 1.98 mumol N,N-dimethylformamide (DMF) hydrolyzed min-1 (mg protein)-1. The native DMFase has a relative molecular mass of 250 000 and is composed of two light-chain (Mr = 15 000) and two heavy-chain (Mr = 105 000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20 degrees C. The activity of the enzyme is inhibited by metal-chelating agents such as EDTA and 2,2'-dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron-containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm. DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40 degrees C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short-chain aliphatic amides such as DMF, N-ethylformamide and N-methylformamide. N,N-dimethylformamide, N,N-dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis-Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver-Burk plot.
The effects of DMSO, ethylene glycol, glycerol, erythritol, dimethylformamide and sucrose on the survival of unfrozen and frozen mouse morulae were examined. All the agents had a deleterious effect on survival of the unfrozen morulae at 20 degrees C. The harmful effect of erythritol was lower at 0 degrees C and sucrose had a protective effect at that temperature. Dimethylformamide and DMSO were more harmful than ethylene glycol and glycerol at both temperatures. Higher proportions of the morulae frozen in DMSO developed into expanded blastocysts when slowly frozen samples were thawed slowly (82%) than when they were thawed rapidly (85%) than when they were thawed slowly (4%). When the samples were frozen in the presence of ethylene glycol or glycerol, higher survival rates were obtained after slow (80--94%) than rapid (1--59%) freezing. When erythritol was used, only embryos thawed rapidly developed (31--50%) in subsequent cultures. Sucrose and dimethylformamide did not afford cryoprotection. Morulae frozen--thawed rapidly in the presence of ethylene glycol, glycerol or erythritol and transferred to recipients developed into normal young.
The present report describes a transformed cell line (AKR-MCA) in which the c-myc proto-oncogene is up-regulated by as much as 14-fold as cultures approach stationary phase growth. The untransformed counterpart AKR-2B cells did not exhibit such an increase in c-myc expression at high cell densities, nor did chemically transformed derivatives of another murine fibroblast cell line (C3H 10T1/2). N,N-Dimethylformamide and retinoic acid reduced c-myc levels in confluent AKR-MCA cells in association with a loss of transformed morphology, a reduction in saturation density, and the formation of a contact-inhibited monolayer at confluency. These findings suggest that the high levels of c-myc in confluent AKR-MCA cells may interfere with the normal signals involved in density-dependent growth regulation in this cell system. The effects of N,N-dimethylformamide and retinoic acid were reversible and dose-related. The half-time for the early, rapid decline in c-myc mRNA was approximately 26 min in response to N,N-dimethylformamide and 38 min in response to retinoic acid, effects which preceded the alterations in morphology and saturation densities. Activation of the latent transforming growth factor-beta in serum-free medium conditioned by confluent AKR-MCA cells, followed by its addition to preconfluent AKR-MCA cells, resulted in an up-regulation of c-myc mRNA. However, addition of serum-containing conditioned medium under similar conditions did not require prior acidification to up-regulate c-myc. Thus, active transforming growth factor-beta may be present in conditioned medium from confluent AKR-MCA cells grown in serum-containing medium, or autocrine factors other than TGF-beta may produce the confluency-associated up-regulation of c-myc and the altered density-dependent growth regulation in AKR-MCA cells.