Isolation and characterization of 5-carbamoylmethyluridine and 5-carbamoylmethyl-2-thiouridine from human urine.

Two new modified uracil nucleosides, 5-carbamoylmethyuridine (ncm5U, I) and 5-carbamoylmethyl-2-thiouridine (ncm5s2U, II) were isolated from a 24 hr collection of a normal human urine. The structures were assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison of the spectral data and HPLC mobilities with those of authentic samples. On the basis of experimental data it appears possible that 5-carbamoylmethyl-2-thio-uridine (ncm5s2U, II) may be a degradation product produced from a labile precursor by the chemical treatments during the isolation procedure. However, the other nucleoside (ncm5U,I) certainly appears to be of metabolic origin and was also found in the urines of one chronic myelogenous leukemia and one lung carcinoma patient. Abbreviations used are: tRNA-transfer ribonucleic acid, TMS-trimethylsilyl, RP-HPLC--reverse phase high performance liquid chromatography, EI--electron impact, cm5U-5-carboxymethyluridine, mcm5U-5-methoxycarbonylmethyluridine, cm5s2U-5-carboxymethyl-2-thiouridine, mcm5s2U-5-methoxycarbonylmethyl-2-thiouridine, t6A-9-beta-D-ribofuranosyl-[N(purin-6-yl)carbamoyl]-1-threonine, C-cytidine, acp3u-3-(3-amino-3-carboxypropyl)uridine, AICR-aminoimidazole carboxamide riboside, alpha-4-PCNR & beta-4-PCNR-9-alpha-D-(or beta-D)-ribofuranosyl-pyridin-4-one-3-carboxamide, H x 7R-7-beta-D-ribofuranosyl hypoxanthine, m3U-3-methyluridine, m1I-1-methylinosine, m1G-1-methylguanosine, DI-5'-deoxyinosine, dms5OA-5'-deoxy-5'-methylthioadenosine sulfoxide, m2(2)G-N2-dimethylguanosine, psi-psi-uridine, A-adenosine, I-inosine, CML-chronic myelogenous leukemia mam5s2U-5-methylaminomethyl-2-thiouridine, ncm5U-5-carbamoylmethyluridine, ncm5s2U-5-carbamoylmethyl-2-thiouridine, UV-ultraviolet, NMR-nuclear magnetic resonance, HPLC-high performance liquid chromatography, GC-MS-gas chromatography-mass spectrometry.

Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine.

The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.

Identification of cis-dichloro-bis-isopropylamine platinum(II) as a major metabolite of iproplatin in humans.

Iproplatin is a quadrivalent second-generation platinum complex undergoing clinical evaluation. In plasma and urine of patients receiving this drug, iproplatin- and platinum-containing metabolites of iproplatin were separated by reverse-phase gradient high performance liquid chromatography. One of the metabolites was identified by cochromatography and electron impact mass spectrometry as cis-dichloro-bisisopropylamineplatinum(II), a metabolite formed by reduction of iproplatin. Incubation of iproplatin with ascorbic acid and cysteine, in vitro, indicates that iproplatin can be easily reduced to cis-dichloro-bis-isopropylamineplatinum(II) by reducing agents. It is hypothesized that the reduction of the quadrivalent complex iproplatin to cis-dichloro-bisisopropylamineplatinum(II) occurs intracellularly.

Metabolism of 1-methyladenosine.

1-[methyl-8-14C] Adenosine was synthesized and its metabolic fate was determined in intact rat. It was found that approximately 57% of 1-[methyl-8-14C] adenosine administered iv was excreted unchanged in the urine and 33% of the excreted radioactivity in the urine was associated with the major metabolite 1-methyl-hypoxanthine and about 4.5% was associated with 1-methylinosine. Very little adenosine or N6-methyladenosine was formed. It is concluded that 1-methyladenosine is initially deaminated by adenosine deaminase to 1-methylinosine which is then cleaved by nucleoside phosphorylase to 1-methylhypoxanthine.

Radioimmunoassay for a novel urinary nucleoside, N6-succinyladenosine.

A sensitive radioimmunoassay has been developed for an unusual urinary nucleoside, N6-succinyladenosine (N6-SAR). Antibodies were raised in rabbits and the antibody specificity was established by binding inhibition radioimmunoassay of compounds with structural similarity to N6-SAR. The assay is sensitive enough to detect up to a nanomole of N6-SAR in 1 ml of urine. The mean N6-SAR urinary excretion value for normal adults was 4.09 mg/gm of creatinine with a standard deviation of 1.22. The measurement of N6-SAR in the urines of a number of subjects with metastatic malignant disease suggests that these patients excreted in their urines elevated amounts of N6-SAR in comparison to that in the urines of normal subjects.

Isolation and characterization of a novel nucleoside, 7-beta-D-ribofuranosylhypoxanthine, from the urine of a chronic myelogenous leukemia patient.

A novel nucleoside, in the amount of 400 micrograms, was isolated from a 24-h collection of urine of a chronic myelogenous leukemia patient. On the basis of ultraviolet, nuclear magnetic resonance, and mass spectrometry and chromatography, its structure was established to be 7-beta-D-ribofuranosylhypoxanthine. The ultraviolet and mass spectral data and the thin layer chromatographic mobilities of the natural material were identical to those of a synthetic sample. High performance liquid chromatographic retention times and the coinjection high performance liquid chromatography of the natural material with the synthetic samples of the alpha and beta-anomers of 7-ribofuranosylhypoxanthines further confirmed the identity of the isolated material as 7-beta-D-ribofuranosylhypoxanthine.

Quantitative aspects of metal ion binding to certain transfer RNA anticodon loop modified nucleosides.

Magnesium and manganese ions bind strongly to the unusual transfer RNA anticodon loop nucleotides, N-[(9-beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]-L-threonine 5'-monophosphate (pt6A) and uridine-5-oxyacetic acid 5'-monophosphate (pV). Potentiometric measurements have shown that the delta G for metal ion-pt6A complex formation is 2-3-times more exothermic than for AMP. Electron-nuclear longitudinal dipolar relaxation data yielded manganese-ligand atom distances which permit a three-dimensional construct of the complex in which metal is coordinated to the phosphate, carboxylate of the threonine side-chain (with the nucleotide in the anti glycosidic conformation) and N7 of the adenine ring. Similarly, manganese binds strongly to pV, involving phosphate and carboxylate functions. It is possible that a facet of the functional role of these unusual residues is to chelate magnesium ions and in so doing permit optimum anticodon loop conformational stability and stability of tRNA-mRNA-ribosome complexes.

On the function of N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine in transfer ribonucleic acid. Metal ion binding studies.

The unusual transfer ribonucleic acid (RNA) anticodon adjacent modified nucleoside N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine, t6A, and its N6-methyl (mt6A) and 5'-phosphate (pt6A) derivatives are efficient ligands for magnesium and manganese ions, as demonstrated by potentiometry and nuclear magnetic resonance spectroscopy. Analysis of the 1H and 13C NMR spectra of t6A in the presence of paramagnetic Mn (II) at 32-34 degrees C has shown that the metal ion binds to the carboxyl group and probably N6 of the side chain as well as the N1 or N7 atom of the adenine ring. A more specific and stronger metal-ligand complex between Mn(II) and pt6A is evident from the 1H, 13C, and 31P NMR data. In this case, the metal forms a complex involving phosphate, N7 of the adenine and the carboxyl group, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparison between them as well as a comparison of the binding between Mg(II) and Mn(II). The stability constants (log K) for 1:1 metal-ligand complexes between Mg(II) and t6A, mt6A, and pt6A are respectively 5.5, 4.3, and 7.1. For Mn(II), the respective values are 6.0, 4.5, and 7.9. In the case of pt6A, the stability constants are about 5 log K units larger than those obtained for Mg(II) and Mn(II) binding to 5'-AMP [Khan, M. M. T., & Martell, A. E. (1967) J. Am. Chem. Soc. 89, 5585]. Thus the threonine side chain is an important determinant in the interaction between the modified nucleosides and metal ions, and these results are supportive of the idea that a facet of the function of this type of unusual nucleoside in transfer RNA is as a specific ligand for magnesium ion, a postulate promulgated earlier [Miller, J. P., Hussain, Z., & Schweizer, M. P. (1976) Nucleic Acids Res. 3, 1185].

Metabolic fate of N6-benzyladenosine and N6-benzyladenosine-5'-phosphate in rats.

The radiolabeled antitumor nucleoside (14C-8)-N6-benzyladenosine and its (14C-8)-5'-phosphate were administered to rats intravenously, and their metabolic fate was studied. Twenty-nine percent of the radioactivity was recovered in the 48-hr urine collection after (14C-8)-N6-benzyladenosine administration. The following metabolites were isolated: unchanged N6-benzyladenosine (20%), adenine (12%), uric acid (5%), and N6-benzyladenine (0.3%). In the case of (14C-8)-N6-benzyladenosine-5'-phosphate, a total of 28% of the radioactivity was recovered in the 48-hr urine collection and the following metabolites were isolated: N6-benzyladenosine (40%), uric acid (12%), adenine (trace), and unidentified urea derivatives (30%). Metabolism of N6-benzyladenosine appears to involve N-debenzylation to some extent, followed by conversion to adenine and uric acid. N6-Benzyladenosine and its 5'-phosphate differ from other adenosine analogs in being retained in significant amounts by the animals.

Synthesis and biological activity of N6-(n-alkylureido)purine ribonucleosides and their 5'-phosphates.

Syntheses and biological activities of 12 N6-(n-alkylureido)purine ribonucleosides (alkyl chain length of 1--10, 16, and 18 carbons) and three N6-(n-alkylureido)purine ribonucleoside 5'-phosphates (chain length of 4, 9, and 10 carbons) are described. The N6-(n-alkylureido)purine ribonucleosides were prepared by a reaction of (2',3',5'-tri-O-acetyl-beta-D-ribofuranosyl)-9H-purine-6-carbamate and n-alkylamine in refluxing pyridine. The 5'-nucleotides were prepared by direct phosphorylation of the corresponding ribonucleoside with phosphorus oxychloride and triethyl phosphate. Some N6-(n-alkylureido)purine ribonucleosides (n-octyl, n-nonyl, and n-decyl) and their nucleotides showed a marked antiproliferative activity against L-1210 cells in culture.

Isolation and characterization of N6-succinyladenosine from human urine.

From the urines of colon carcinoma patients and normal subjects we have isolated a nucleoside in which an amino group of aspartic acid is attached to the six position of purine ribonucleoside. The structure, N6-succinyladenosine, N-(9-B-D-ribofuranosylpurin-6-yl)aspartic acid was assigned on the basis of spectral data, chemical degradation, and by synthesis. The ultraviolet and mass spectra, chromatographic and electrophoretic mobilities, and the chemical properties of the naturally occurring nucleoside were identical to those of the synthetic N6-succinyladenosine. In contrast to the methylated and hypermodified nucleosides which are products of RNA catabolism, this urinary nucleoside appears to be derived from adenylosuccinic acid, a key intermediate required in the biosynthesis of ubiquitous, natural purine nucleotide adenosine-5'-monophosphate (AMP).

Synthesis and biological activities of some N4-substituted 4-aminopyrazolo(3,4-d)pyrimidines.

Syntheses and biological activities of 26 N4-substituted 4-aminopyrazolo[3,4-d]pyrimidines as analogs of naturally occurring modified nucleic acid bases, N-(purin-6-ylcarbamoyl)-L-threonine and N6-(delta2-isopentenyl)adenine, are described. 4-Aminopyrazolo[3,4-d]pyrimidine was converted into the desired intermediate, ethyl pyrazolo[3,4-d]pyrimidine-4-carbamate (2). 4-Ureidopyrazolo[3,4-d]pyrimidines (6-26) were prepared by displacement of the ethoxy group of the carbamate 2 by amino acids and a variety of amines and by a reaction of 4-aminopyrazolo[3,4-d]pyrimidine (1) with isocyanates. N4-Alkylaminopyrazolo[3,4-d]pyrimidines were generally prepared by displacement of the chlorine from 4-chloropyrazolo[3,4-d]pyrimidine with various amines. Several analogs exhibited moderate to very good growth inhibitory activities against cultured L1210 leukemia and 6410 human leukemic myeloblasts.

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.