Synthesis of guanosine and its derivatives from AICA-riboside

A novel and convenient method for the synthesis of guanosine is described. The reaction of AICA-riboside with sodium methylxanthate gave 2-mercaptoinosine in almost quantitative yield. The latter was oxidized with hydrogen peroxide to afford inosine-2-sulfonic acids, which was readily animated to give guanosine in excellent yield. Similarly, the preparation of N²-methylguanosine and N²,N²-dimethylguanosine, minor constituents of transfer RNA, was also accomplished. Furthermore, this procedure was extended to the synthesis of 2′,3′-O-isopropylideneguanosine and the isopropylidene derivatives of various N²-substituted guanosines from 2′,3′-O-isopropylidene-AICA-riboside. Guanosine via 2′,3′-O-isopropylideneguanosine was successfully phosphorylated to give 5′-guanylic acid.     

Complexing of 3d transition metal ions with 9-substituted purines. IV. The effect of base-stacking on the basicity and complexing-ability

A number of 9-methylpurines were equilibrated between carbon tetrachloride or chloroform and aqueous solutions containing either nickel(II) perchlorate or N⁶,N⁶-dimethyladenosine. The equilibrium constants for the complex formation with nickel(II) ion and association with N⁶,N⁶-dimethyladenosine were calculated on the basis of the distribution data. The results, together with those obtained in the presence of both nickel(II) perchlorate and N⁶,N⁶-dimethyladenosine, are interpreted to indicate that stacking-association with the latter compound reduces the complexing-ability of 9-methylpurines. The protonation of guanosine in the presence of caffeine was examined potentiostatically and its association with caffeine studied by phase-solubility measurements. Association with caffeine was shown to lower the basicity of guanosine.     

Resolution of partition coefficients in the transverse plane of the lipid bilayer

The distribution of a small lipid soluble molecule across a lipid bilayer has been determined using fluorescence quenching techniques. The neutral form of the amine, N,N-dimethylaniline (DMA) quenches the fluorescence of a series of n-(9-anthroyloxy) fatty acids (n = 2,6,9,12,16) which place a fluorophore at a graded series of positions from the surface to the centre of the lipid bilayer. A method is described for determining the partition coefficient of a quencher at each transverse position. The results show that DMA is located at all depths within the bilayer leaflet but that it is concentrated at the bilayer centre and to a lesser extent at the bilayer surface.     

High-performance liquid chromatography separation of some NAD+ derivatives suitable for covalent binding

Separation of NAD⁺, N¹-carboxymethyl-NAD⁺, N⁶-carboxymethyl-NAD⁺, and N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ by high performance liquid chromatography is described. Reversed-phase chromatography with the acidic mobile phase (phosphate buffer pH 2.0–3.6) proved to be the most suitable method, particularly for the separation of impurities. The proposed method can be used for monitoring the course of the synthesis of N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ and for the separation of the intermediates. Identification of the peaks was performed by means of spectroscopic measurement as well as a specific coenzyme activity test. Performance of the described method is greater in comparison with thin-layer chromatography.     

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps.The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD⁺, cyclic AMP, NADP⁺ and uridinediphosphoglucose (UDPG).In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.     

Conversions of the glucocorticoid · receptor complex of rat thymocyte cytosol, studied by partition in an aqueous dextra-polyethylene glycol two-phase system

Tritiated glucocorticoid · receptor complex was formed by incubation of [³H] triamcinalone acetonide at –5°C with thymocyte cytosol, made 40% with respect to glycerol. The cytosol was then incubated at various temperatures, ionic strengths and cytosol concentrations for various times. At the end of the incubations, the partition coefficient of the complex in an aqueous dextranpolyethylene glycol two-phase system was determined. By the criterion of time-dependent changes of the partition coefficient, two differential conversions of the complex were distinguished: 1. A rapid, ionic strength-dependent process, increasing the partition coefficient. 2. A slower, temperature-, ionic strenght- and cytosol concentration-dependent process, increasing the partition coefficient.The effect of the salt composition of the partition mixture on the partition coefficient indicated, that the complex has a pI below 6. The effect of pH of the partition mixture indicated that the conformation of the state of aggregation of the complex is dependent on pH.     

Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates

The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO₂ under acidic conditions, N⁵-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N²,N²-dimethylguanosine generated no product. In a similar experiment without NO₂^–, participation of a Dimroth rearrangement was ruled out. In the guanosine–HNO₂ reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20°C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by HNO₂ is proposed.     

Reactivity-selectivity properties of reactions of carcinogenic electrophiles and nucleosides: Influence of pH on site selectivity

6-Chloromethylbenzo[a]pyrene (6-CMBP) labeled with ¹³C in the chloromethyl group was used as a model for those carcinogens which form essentially free carbocations. Using ¹³C-NMR to identify products, the selectivity with which this electrophile modifies nucleosides was investigated. At pH 7, guanosine and deoxyguanosine are the most nucleophilic nucleosides toward the carbocation generated by solvolysis of 6-CMBP. Attack at N-7 predominates over attack at N-2. At higher pH, the nucleophilicity of guanosine and deoxyguanosines increases markedly. In addition, the site of modification changes to N-1 with secondary modification at O-6. The pH dependence of the rate of this reaction implicates a group with pK-value approx. 8.7 which was assigned to the hydrogen on N-1. The presence of a methyl group on the N-7 position of guanosine lowers this pK-value to approx. 7.2. Consequently, N⁷-methylguanosine shows the high nucleophilicity at physiological pH that guanosine has at high pH. These observations lead to the suggestion of a one base: two-site model for chemical carcinogenesis.     

N-substituted maleimide inactivation of the response to taste cell stimulation

N-Ethylmaleimide (NEM) irreversibly inactivates the response of gustatory cells to stimulation by NaCl, sucrose and hydrogen ions. The rate of inactivation can be measured by monitoring the decay of NaCl-stimulated summated electrophysiological activity at the chorda tympani nerve in the presence of NEM. The observed pseudo first-order rate constants are linear with NEM concentration, and the second-order rate constant is 0.38 M^?1 sec^?1. Other N-substituted maleimides, such as N-methylmaleimide and N-butylmaleimide, which have ether:water partition coefficients similar to NEM, inactivate the NaCl-stimulated response at rates comparable to NEM. However, the hydrophobic derivative, 4-(N-maleimido)phenyltrimethylammonium has a significantly lower ether:water partition coefficient and is essentially ineffective as an inactivator of the NaCl response. These results, together with the observation that the inactivation rate is independent of pH between 4.5 and 7.0, indicate the inactivation site is either intracellular or buried within the cell membrane at a locus inaccessible to most extracellular fluids. The rate of inactivation of the sucrose and HCl responses were measured indirectly and found to be comparable to the NaCl-stimulated inactivation rate, indicating the inhibited event is common to the transduction of the response for all of the stimuli examined. Possible sites of inactivation by N-Substituted maleimides are considered in the context of the results. This kinetic approach should have application in searching for and characterizing receptor-specific as well as other classes of taste cell inhibitors.     

Preparative solvent partition chromatography of butyrylated cyclic AMP analogues

A simple and effective separation of cyclic adenosine-3′,5′-monophosphate (cAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5 m ammonium acetate (4:1) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated cAMP's purified by this technique. In addition, it has analytical utility in that it allows complete separation of N⁶-monobutyryl cAMP from O^2′-monobutyryl cAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (N⁶,O^2′-dibutyryl cAMP), and this is demonstrated under several conditions.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The ¹³C resonances of N^α,N-[¹³C]dimethylserine of partially ¹³C reductively methylated glycophorin A^M were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the ¹³C resonances of N^α,N- [¹³C]dimethylserine of fully ¹³C reductively methylated deglycosylated glycophorin A^M. The ‘major’ component of N^α,N- [¹³C]dimethylserine of glycophorin A^M did not titrate, whereas the ‘minor’ component titrated with a pK_a of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the N^α,N- [¹³C]dimethylserine resonances of ¹³C reductively methylated deglycosylated glycophorin A^M.     

Synthesis,FT-IR and 1H NMR studies of alkali and alkaline earth metal complexes with guanosine

The synthesis of complexes of Li(I), K(I), Mg(II), Ca(II) and Ba(II) with guanosine in basic non aqueous solutions is described. The complexes were of two types: (1) complexes having the general formula, M(Guo)_nX_m·YH₂O·ZC₂H₅OH, where M = Mg(II), Ca(II), Ba(II) and Li(I), n = 1,2,4, X = Cl^?, Br^?, NO₃^?, ClO₄^? and OH^?, m = 1,2, Y = 0?6 and X = 0?2, and (2) complexes with the general formula, M(GuoH-1)(OH)_n^?1·YH₂O, where M = K(I), Ca(Il) and Ba(II), GuoH-1 =Ionized guanosine at N₁, n = 1,2 and Y = 1?3. The complexes are characterized by their proton nuclear magnetic resonance (¹H NMR) and Fourier transform infrared (FT-IR) spectra. The FT-IR and ¹H NMR data of the non ionized nucleoside complexes suggest that the metal binding is through the N₇-site of guanine and that the anion (X) is hydrogen bonded to N₁H and NH₂ groups. In the N₁-ionized guanosine complexes the metal binding is via the O₆^? of guanine. All the complexes formed exhibited a transition of the sugar conformation from C₂′-endo/anti in the free nucleoside to C₃′-endo/anti in the metal complexes.     

Modification of ribonucleic acid by chemical carcinogens. VI. Effect of N-2-acetylaminofluorene modification of guanosine on the codon function of adjacent nucleosides in oligonucleotides

Oligonucleotides containing a guanosine residue on the 5′ or the 3′ side of tri- and tetranucleotides were prepared. The guanosine residue was modified with the chemical carcinogen N-2-acetylaminofluorene and the control and modified oligonucleotides were tested for their ability to stimulate ¹⁴C-labeled amino-acyl-tRNA binding to ribosomes. The effects of the modification are twofold. The first is that if the guanosine residue to which the drug is eovalently bound is part of a codon the oligonucleotide is completely inactive in the ribosomal binding assay. The second is that if an adenosine residue is adjacent to either the 5′ or 3′ side of the modified guanosine, as in (Ap)₃G or G(pA)₃, there is partial inhibition of ¹⁴C-labeled lysyl-tRNA binding to ribosomes. This inhibitory effect extends only to the function of the immediately adjacent adenosine since the chemical modification of guanosine residues in (Ap)₄G or G(pA)₄ did not impair their ability to code for lysine. In contrast to these findings if there is a uridine residue adjacent to the modified guanosine, as in (Up)₃G or G(pU)₃ there is no effect on ¹⁴C-labeled phenylalanyl-tRNA binding to ribosomes. Proton magnetic resonance spectra of UpG, GpU and the corresponding dinners in which the guanosine residue was modified with the drug failed to indicate a stacking interaction between the fluorene moiety and the adjacent uridine residue. This is in contrast to previous studies demonstrating a strong stacking interaction between fluorene and adjacent adenosine residues. Taken together these results indicate that acetylaminofluorene modification of guanosine next to an adenosine residue in oligonucleotide inhibits its ribosomal binding capacity. The stacking interaction with adjacent adenosine, and not with adjacent uridine residues, in oligonucleotides probably accounts for the effects observed in the ribosomal binding assay. These data are consistent with our previously described “base displacement” model.     

Studies on the Guanosine Degrading System in Bacterial Cell

A highly purified preparation of the enzyme, guanosine deaminase, has been obtained by a four-step purification procedure from the cells of Pseudomonas convexa No. 149. The enzyme deaminates guanosine, deoxyguanosine and 8-azaguanosine, and the deamination of guanosine is inhibited by Hg²⁺ and 4-amino-5-imidazole carboxamide ribonucleoside.

The enzyme has the optimum pH at pH range from 6.0 to 6.5 and the optimum temperature at 45°C. Metal ions are not required for the enzyme activity.

The molecular weight of the enzyme is 1 × 10⁵ to 2 × 10⁵.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Tracer Determinations of Human Red Cell Membrane Permeability to Small Nonelectrolytes

A flow system has been used to determine the permeability of human red cell membranes to four small nonelectrolytes labeled with ¹⁴C. The permeability coefficients, ω, in units of mol dyne^-1 sec^-1 x 10¹⁵, are: ethylene glycol, 6; urea, 13; formamide, 22; and methanol, 131. The values for urea and formamide are in good agreement with values obtained by Sha'afi, Gary-Bobo, and Solomon by the minimum method. The unusually high value for ω for methanol is ascribed to its solubility in the red cell membrane since its ether: water partition coefficient is 0.14, higher by more than an order of magnitude than the ether: water partition coefficient for water. The other three solutes are hydrophilic and are characterized by values of ω which behave consistently with those of other hydrophilic amides and ureas. The values of ω for the three hydrophilic solutes measured are also consistent with an equivalent pore radius of about 3.5 A in agreement with previous estimates made on the basis of other types of studies.     

Determination of three different pools of reduced one-carbon-substituted folates: I. A study of the fundamental chemical reactions

The reduced one-carbon-substituted derivatives of folic acid can be grouped in three pools according to their response to acid treatment. Pool 1 is made up of N⁵,N¹⁰-methylene-tetrahydrofolic acid and unsubstituted dihydro- and tetrahydrofolic acid which at pH 1.0 and subsequent exposure to air cleave to p-aminobenzoylglutamic acid. Pool 2 is made up by the acid-stable N⁵-methyl-tetrahydrofolic acid, and pool 3 includes N⁵,N¹⁰-methenyl-tetrahydrofolic acid, N¹⁰-formyltetrahydrofolic acid, N⁵-formyltetrahydrofolic acid, and N⁵-formiminotetrahydrofolic acid, all of which convert to the stable N⁵,N¹⁰-methenyl-tetrahydro form when acid treated. Conditions are described to selectively cleave the C⁹-N¹⁰ bond of the folates of pool 1, pools 1 + 2, and pools 1 + 2 + 3. The cleaved pools are quantitated as the Bratton-Marshall azo dyes of p-aminobenzoylglutamate. The uncleaved pools are converted to Bratton-Marshall-negative products. Pool 1 is determined by converting pool 2 to 4a-hydroxy-5-methyltetrahydrofolic acid and pool 3 to N¹⁰-formylfolic acid, both Bratton-Marshall negative, by 10% hydrogen peroxide oxidation at pH 6.0. Pools 1 + 2 are cleaved with 0.015% hydrogen peroxide and 0.1% potassium permanganate at pH 9.0 which convert the N⁵-methyltetrahydrofolic acid to the acid-cleavable N⁵-methyl-dihydrofolic acid. Pool 3 oxidizes to the Bratton-Marshall-negative N¹⁰-formylfolic acid. Pools 1 + 2 + 3 are cleaved by first reducing pool 3 to N⁵-methyltetrahydrofolic acid with sodium borohydride followed by oxidation at pH 9.0 to its acid-labile dihydro form. Determination of the poly-γ-glutamyl chain length of each pool is possible by chromatographing the azo-p-aminobenzoylpolyglutamates with authentic synthetic markers.     

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry

A method for the quantification of acetylpolyamines, N¹,N¹²-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N¹,N⁸-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using ¹⁵N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of α,ω-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N¹-acetylspermidine and N⁸-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).     

Examination of Haldane's first law for the partition of CO and O2 to hemoglobin A0

A study of the oxygen replacement reaction of carbon monoxide-saturated hemoglobin (HbA₀) was carried out using spectroscopic, calorimetric, and pH titration methods. Under fully saturated conditions the replacement reaction can be defined by a single partition constant over all ratios of bound oxygen to carbon monoxide. This indicates that under saturating conditions Haldane's first law for the ligand binding of gas mixtures holds for any CO/O₂ ratio. It further shows that there is no appreciable difference in relative CO-O₂ affinity between the α- and β-chains. The same partition coefficient was found to hold for different pH, buffer, and allosteric effector conditions. The lack of any pH dependence of the partition coefficient was confirmed by the absence of proton changes for the replacement reaction. The temperature dependence of the partition coefficient and calorimetric results yield a value for the enthalpy of the reaction of ?3.65 ± 0.29 kcal/mol/heme.