Inosine preparation

As described in U.S. Patent 3,049,536, inosine may be prepared starting with adenosine. 

By this method adenosine triphosphate (ATP) and inosine triphosphate (ITP) were prepared in 89% and 93% yield, respectively 51]... 

Tipson devoted most of his years in Levene s laboratory accomplishing seminal work on the components of nucleic acids. To determine the ring forms of the ribose component of the ribonucleosides he applied Haworth s methylation technique and established the furanoid structure for the sugar in adenosine, guanosine, uridine, and thymidine. He showed that formation of a monotrityl ether is not a reliable proof for the presence of a primary alcohol group in a nucleoside, whereas a tosyl ester that is readily displaced by iodide affords clear evidence that the ester is at the 5-position of the pentofuranose. Acetonation of ribonucleosides was shown to give the 2, 3 -C -isopropyl-idene derivatives, which were to become extensively used in nucleoside and nucleotide chemistry, and were utilized by Tipson in the first chemical preparation of a ribonucleotide, inosinic acid. 

Concerning human studies, only three reports have been reported (for review see Burnstock et al., 2000). Bleehen and Keele (1977) reported observations on the algogenic actions of adenosine compounds on blister base preparations. Coutts et al. (1981) injected ATP, ADP, AMP, adenosine, adenine and inosine intradermally. The area of erythema induced by the injection was delineated at 30 s. and again after a further 4.5 min when the size of the response was maximal. ATP, ADP and AMP evoked weal and flare responses in the skin in a dose-dependent manner. The rank order of potency was ATP > ADP > AMP other metabolites were apparently inactive. Injections of ATP and high doses of ADP produced a sensation of persistent pain. 

Mixtures of photolabile 2 - and 3 -0-(2-nitrobenzyl)ribonucleosides were obtained in good yields when adenosine, uridine, cytidine, and inosine were treated with 2-nitrophenyldiazomethane in the presence of tin(II)chloride [362]. The dibutylstan-nylene approach has been used for the preparation of 2 -(4-nitrobenzyl)uridine [366]. 2-Nitrobenzyl chloride and sodium hydride in DMF gave the 2 -ethers in 26-37% yield, depending on the starting nucleoside [363, 367]. 

Figure 8.8 Retention times for adenosine (22.45 pmol), inosine (22.37 pmol), and hypoxan-thine (44.08 pmol) (A) standard prepared in mobile phase (B) standard prepared in incubation fluid or cell suspension medium. Conditions mobile phase, 125 mM potassium dihydrogen phosphate, 1.5% (v/v) acetonitrile, 20 mM triethylamine, and 1.0 mM tetrabutylammonium hydrogen sulfate (TBAHS) (pH 6.5) flow rate, 0.5 ml/min. (Reprinted from Ref. 18 with permission.)...

Reagents. Cyclic nucleotides (3, 5 -cyclic adenosine monophosphate (c-AMP), 3, 5 -cyclic guanosine monophosphate (c-GMP), and 3, 5 -cyclic inosine monophosphate (c-IMP)) sodium tetraborate hydrochloric acid and potassium hydroxide were purchased from Sigma Chemical Company, St. Louis, Missouri). Millex disposable filter units (0.22 pm) were obtained from Millipore Corporation (Bedford, Massachusetts). Triply distilled and deionized water was used for the preparation of buffer solutions. Both buffers and samples were routinely degassed with helium after filtration (using microfilter units). 

Cyclic acetals of the naturally occurring N-ribofuranosides have been prepared. Levene and Tipson156 found that inosine will condense with acetone in the presence of zinc chloride to give a monoisopropylidene derivative. Since this compound afforded a monotosyl ester in which the tosyloxy group was readily replaced by iodine, it was assigned structure CYI. Phosphorylation of CYI followed by hydrolytic removal... 

Assay Transfer about 500 mg of sample, accurately weighed, into a 1000-mL volumetric flask, dissolve in and dilute to volume with 0.01 N hydrochloric acid, and mix. Transfer 10.0 mL of this solution into a 250-mL volumetric flask, dilute to volume with 0.01 N hydrochloric acid, and mix. Using a suitable spectrophotometer and 0.01 N hydrochloric acid as the blank, determine the absorbance of this solution and of a similarly prepared solution of USP Disodium Inosinate Reference Standard in 1-cm cells with the maximum at about 250 nm. Calculate the quantity, in milligrams, of CioHnN4Na2OgP in the sample taken by the formula... 

Figure 2.10 The use of enzymes to identify an unknown compound. The compound tentatively identified as IMP, based on its retention time of 2 minutes (chromatogram obtained at zero reaction time), was incubated with a commercially available preparation of S -nucleotidase. Samples of the incubation mixture were removed and analyzed by HPLG The chromatograms, obtained at 10 and 20 minutes of reaction time, showed a reduction in the area of the IMP peak and an increase in the area of the inosine (Ino) peak, confirming that the original peak was IMP.

The possibility that more of the reaction product Ado had remained on the column was ruled out by using different mobile phases to elute all bound material. The chromatograms accounted for all the products. Since we had not expected any side reactions, we quantitated the yield of reaction products on the basis of area, assuming the presence of only adenosine-containing compounds. The formation of inosine, with a 50% reduction in extinction coefficient, could account for the apparent lack of recovery. Therefore, we considered the presence of secondary reactions. Either AMP had been converted to IMP, or adenosine was converted to inosine (Ino). By comparing the retention time of the third peak to authentic standards, we ruled out IMP as a product, and thus the identity of peak 3 was established as inosine. This led us to conclude that the commercial preparation of alkaline phosphatase was contaminated with a second activity, adenosine deaminase. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

Figure 10.13 HPLC of FoA and its metabolites produced by activities present in an S-30 fraction prepared from rat liver FoB is a formycin analog of inosine. Operating conditions mobile phase, 0.1 M KH2P04 adjusted to pH 5.5 with NaOH with 10% methanol flow rate, 2 mL/min room temperature Qg /xBondapak column packing fluorescence, excitation at 300 nm, emission about 320 nm. (A) Chromatogram obtained with standards as indicated at approximately 10 /xg of each. Sensitivity of fluorescence detection, 0.5 AUFS units full scale. (B) Chromatogram obtained immediately after addition of S-30 to a reaction mixture containing FoA and ATP. (C and D) Chromatograms obtained after 8 and 16 minutes of incubation, respectively. [From Dye and Rossomando, 1982. Reprinted by permission from Bioscience Reports, 2 229-234 (1982).]...

The technique of ultrafiltration for serum sample preparation has been studied by several investigators (H7, HIO, K9, S19, V4, V5). In this technique the serum sample is passed through an exclusion membrane which exhibits a 95% retention for compounds with MW > 25,000. Thus, low-molecular-weight constituents pass through the membrane, whereas proteins or protein-bound constituents are retained. Hartwick ct al. (HIO) reported excellent recoveries for xanthosine, inosine, guanosine, theobromine, theophylline, and caffeine. Tryptophan showed poor quantitative recovery in accordance with the findings of previous investigators (K33, 01). 

Base-induced (KH or /-BuOK) cyclizations of o-alkynylanilines were utilized to prepare 2-substituted indoles and poly-substituted indoles. For example, treatment of alkynes 86 with KH gave the corresponding indoles 87 <03T1571>. Similar base-mediated cyclizations and related indole syntheses were utilized to prepare indole inhibitors of 5 -inosine monophosphate dehydrogenase <03BMCL1273>. Moreover, base-induced cyclizations of arylacetonitriles with oxalic acid bis(imidoyl)chlorides provide a route to 2-alkylidene-3-iminoindoles <03CEJ3951>. 

Similar methods have been used to prepare nucleosides including inosine (62JCS2937) which results from the l-/3-D-ribofuranosylimidazole (304 Scheme 114) and in a similar manner, 7-/3-D-ribofuranosylhypoxanthine (306) was obtained from the isomeric aminoimidazole nucleoside (307 Scheme 115) (59JCS2893). 1-Benzylinosine has also been prepared by cyclization of the aminoimidazolebenzylamide (305) with cold aqueous alkali (58JA3899). In like manner, 9-methyl- and 8,9-dimethyl-6-thioxo-l,6-dihydropurine (308)... 

Chloro-9-(/3-D-ribofuranosyl)purine has similarly been prepared from inosine (63JOC945) by chlorination of 6-thioinosine (63MI40903) or, perhaps most successfully, by reaction of 2, 3, 5 -tri-0-acetylinosine with (chloromethylene)dimethylammonium chloride when yields of 70% are produced (B-78MI40903, p. 611). The latter reagent is obtained from phosgene and DMF in chloroform solution . 

Moreover, [7- N]-labeled 2 -deo.xyadenosine, -inosine. and -guanosine have been prepared. For this purpose, pyrimidine-4.6-diamine or pyrimidine-2,4,6-triamine was reacted... 

Amino-l-0 -o-ribofuranosyl)imidazole-4-carboxamide (5.16 g, 20 mmol) and hexachloroethane (40 g, 100 mmol) were added to a solution of raethanolic NaOMe (prepared from 7.8 g of metallic Na and 150 mL of MeOH), and the solution was heated to reflux for 3 h with stirring. Paper chromatography of this solution showed two spots which, on elution, corresponded spectrophotometrically to the starting AICA-riboside and inosine (I). HjO (100 mL) was added, the hexachloroethane was removed by filtration and the filtrate was neutralized to pH 7 by adding portionwise Amberlite IR-120 (IH form). The resin was removed by filtration and washed with ILO. The combined filtrate and washings were concentrated in vacuo to give a crystalline mass, which was rccrystallizcd (HjO) to afford a pure sample yield 3 g (51 %). 

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