Inosine Derivatives Research Articles

Selective Methylation of Nucleosides via an In Situ Generated Methyl Oxonium.

A very mild and efficient procedure has been developed for the preparation of N-methylated uridine, pseudouridine, guanosine and inosine derivatives. This process was compatible with free hydroxyls within the ribose and did not require precautions on the protection or deprotection of other functionalities. The key to this extremely mild methylation without protection relied on the in situ generated methyl oxonium from the Wittig reagent and methanol. A putative mechanism for the selective methylation was also proposed.

Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance.

Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.

Ascertaining the activity and inhibition of adenosine deaminase via fluorescence-based assays.

A fluorescence-based assay for adenosine deaminase (ADA) activity and inhibition, which may also be formatted as an inhibitor discovery assay, is described. It relies on differences in fluorescence between an isothiazolo-based adenosine analogs (tzA) and its deaminated product, the corresponding inosine derivative (tzI), which facilitates a real-time monitoring of enzymatic activity. Inhibitors are added to the enzyme-substrate reaction mixture at various concentrations and the fluorescence signal is recorded over 10min. The percent inhibition is calculated from the signal change at 10min relative to the uninhibited reaction. The percent inhibition is plotted against inhibitor concentration and fitted to a Hill curve. IC50 values are then calculated.

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Comparison of the diastereoisomeric excess of uridine, inosine and adenosine cyanohydrins determined by HPLC-DAD and 1H NMR.

ABSTRACTThe separation of the diastereoisomers of the nucleoside derivatives of uridine, inosine and adenosine was performed by HPLC using chiral and no chiral columns, it was observed with the no chiral columns the resolution was good enough to determine diastereoisomeric excess. These methods were compared with 1H NMR, and no significant differences were observed between the three techniques. Diastereoisomeric uridine (3a), inosine (3b) and adenosine (4c) cyanohydrins were resolved by 1H nuclear magnetic resonance (1H NMR), chiral normal phase-high-performance liquid chromatography-diode array detector (NP-HPLC-DAD) and reversed phase (RP-HPLC-DAD); these methods allowed the assesment of the percent diastereoisomeric excess (% de) of the nucleosidic cyanohydrins of 3a (4, 6 and 4), 3b (10, 8 and 6) and 4c (4, 4 and 4). To the best of our knowledge, there are no reports using analytical techniques for the separation of the epimers of 3a, 3b and 4c.

Synthesis of cyclic N (1)-pentylinosine phosphate, a new structurally reduced cADPR analogue with calcium-mobilizing activity on PC12 cells.

Cyclic N1-pentylinosine monophosphate (cpIMP), a novel simplified inosine derivative of cyclic ADP-ribose (cADPR) in which the N1-pentyl chain and the monophosphate group replace the northern ribose and the pyrophosphate moieties, respectively, was synthesized. The role played by the position of the phosphate group in the key cyclization step, which consists in the formation of a phosphodiester bond, was thoroughly investigated. We have also examined the influence of the phosphate bridge on the ability of cpIMP to mobilize Ca2+ in PC12 neuronal cells in comparison with the pyrophosphate bridge present in the cyclic N1-pentylinosine diphosphate analogue (cpIDP) previously synthesized in our laboratories. The preliminary biological tests indicated that cpIMP and cpIDP induce a rapid increase of intracellular Ca2+ concentration in PC12 neuronal cells.

Metabolite Signatures in Hydrophilic Extracts of Mouse Lungs Exposed to Cigarette Smoke Revealed by 1H NMR Metabolomics Investigation.

1H-NMR metabolomics was used to investigate the changes of metabolites in the lungs of mice with and without being exposed to a controlled amount of cigarette smoke. It was found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine were significantly changed in the lungs of mice exposed to cigarette smoke when compared with controls regardless the mice were obese or of regular weight. The decreased ATP, ADP, AMP and elevated inosine suggested that the deaminases in charge of adenosine derivatives to inosine derivatives conversion would be significantly changed in the lungs of mice exposed to cigarette smoke. Indeed, transcriptional study confirmed that the concentrations of adenosine monophosphate deaminase 2 and adenosine deaminase 2 were significantly changed in the lungs of mice exposed to cigarette smoke. We also found that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) was significantly increased in the lungs of obese mice compared with those of the regular weight mice. The GPC/PC ratio was further elevated in the lungs of obese group exposed to cigarette smoke.

Synthesis of inosine 6-phosphate diesters via phosphitylation of the carbonyl oxygen

Inosine derivatives bearing a phosphodiester group at the O(6)-position of the nucleobase were synthesized via phosphitylation of the carbonyl oxygen using phosphoramidites activated by non-nucleophilic acidic activators.

The NMR study of derivatives of substituted inosine – The precursors of AICAr

In this article we describe how the simultaneous presence and interaction of the aromatic ring at position 1 of an inosine derivative with the acetyl group at positions 2, 3, 5 of the furanose ring convert conformation of the nucleoside into the major conformer in solution. During the synthesis of AICAr, we obtained products ( A and B ) of inosine protected with 2,4-dinitrochlorobenzene, with two sets of NMR signals, each with very similar chemical shifts. Analysis of experimental NMR spectra and theoretical GIAO – DFT calculations were performed to obtain information about the stereochemistry of the products.

ChemInform Abstract: Synthesis and Antiviral Activity of Hydrophosphorylated Inosine Derivatives.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Intramolecular Cation−π Interactions As the Driving Force To Restrict the Conformation of Certain Nucleosides

Despite the well-established importance of intermolecular cation-pi interactions in molecular recognition, intramolecular cation-pi interactions have been less studied. Here we describe how the simultaneous presence of an aromatic ring at the 5'-position of an inosine derivative and a positively charged imidazolium ring in the purine base drive the conformation of the nucleoside toward a very major conformer in solution that is stabilized by an intramolecular cation-pi interaction. Therefore, the cation-pi interaction between imidazolium ions and aromatic rings can also be proposed in the design of small molecules where this type of interaction is desirable. The imidazolium ion can be obtained by a simple acidification of the pH of the media. So a simple change in pH can shift the conformational equilibrium from a random to a restricted conformation stabilized by an intramolecular cation-pi interaction. Thus the here described nucleosides can be considered as a new class of pH-dependent conformationally switchable molecules.

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′- O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5′- O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5′- O-tritylinosine on human TP. A cavity in which 5′- O-tritylinosine could fit was identified in the vicinity of the Gly405–Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency ( V max/ K m of mutant enzyme ∼50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5′- O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.

O6‐(Benzotriazol‐1‐yl)inosine Derivatives for C6 Modification of Purine Nucleosides

A new class of reactive nucleosides, O(6)-(benzotriazol-1-yl) derivatives of inosine and 2'-deoxyinosine, have been developed via reaction of silyl-protected or unprotected inosine and 2'-deoxyinosine with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). Alternatively, the silyl-protected O(6)-(benzotriazol-1-yl) derivatives can be synthesized via reaction of protected inosine and 2'-deoxyinosine with triphenylphosphine/iodine/1-hydroxybenzotriazole. These new O(6)-(benzotriazol-1-yl) inosine derivatives are excellent reagents for the synthesis of other nucleoside analogues via S(N)Ar reaction with a range of nucleophiles.

Synthetic Utility of an Isolable Nucleoside Phosphonium Salt

The reaction of O6-benzyl-3',5'-bis- O-( tert-butyldimethylsilyl)-2'-deoxyxanthosine with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) yielded the nucleoside C-2 tris(dimethylamino)phosphonium hexafluorophosphate salt as a stable, isolable species. This is in contrast to reactions of inosine nucleosides with BOP, where the in situ formed phosphonium salts undergo subsequent reaction to yield O6-(benzotriazol-1-yl)inosine derivatives. The phosphonium salt obtained from the 2'-deoxyxanthosine derivative can be effectively used to synthesize N2-modified 2'-deoxyguanosine analogues. Using this salt, a new synthesis of an acrolein-2'-deoxyguanosine adduct has also been accomplished.

Synthesis of 4- N-alkyl and ribose-modified AICAR analogues on solid support

Herein, we report the solid-phase synthesis of several 5-aminoimidazole-4-( N-alkyl)carboxamide-1-ribosides (4- N-alkyl AICARs) and the corresponding 2′,3′-secoriboside derivatives. The method uses the N-1-dinitrophenyl-inosine 5′-bonded to a solid support. This inosine derivative has the C-2 of the purine base strongly activated towards the attack of N-nucleophiles thus allowing the preparation of several N-1 alkylated inosine supports from which a small library of 4- N-alkyl AICAR derivatives has been synthesized. A set of new 4- N-alkyl AICA-2′,3′-secoriboside derivatives have also been obtained in high yields by solid-phase cleavage of the 2′,3′-ribose bond.

A Novel Polymer Supported Approach to Nucleoside Modification

Polymer-supported O(6)-(benzotriazol-1-yl)inosine derivatives (Pol-I and Pol-dI) have been synthesized reasonably effectively via reaction of nucleoside phosphonium salts with polymer-linked HOBt (Pol-HOBt). In constast to solution chemistry, use of polymer-supported BOP (Pol-BOP) did not lead to efficient nucleoside loading. Presence of the nucleosides on the support could be readily detected by MAS NMR. Exposure of the polymer-supported nucleosides, Pol-I and Pol-dI, to alcohol, phenol, thiol and amine nucleophiles caused cleavage from the support leading directly to the C-6 modified nucleoside analogues. To our knowledge, these are the first examples of the application of such technology for nucleoside modification. Where possible, results of reactions with the polymer-supported nucleosides are compared to those from solution chemistry, providing insight into the differences between the two techniques. These new polymer-supported nucleosides can be conveniently utilized for diversity-oriented synthesis.

Unusual Deoxygenation and Reactivity Studies Related to O6-(Benzotriazol-1-yl)inosine Derivatives

New and unusual developments related to the chemistry of O6-(benzotriazol-1-yl)inosine derivatives are reported. First, a simple, scalable method for their syntheses via the use of PPh3/I2/HOBt has been developed and has been mechanistically investigated by 31P(1H) NMR. Studies were then conducted into a unique oxygen transfer reaction between O6-(benzotriazol-1-yl)inosine nucleosides and bis(pinacolato)diboron (pinB-Bpin) leading to the formation of C-6 (benzotriazol-1-yl)purine nucleoside derivatives and pinB-O-Bpin. This reaction has been investigated by 11B(1H) NMR and compared to pinB-O-Bpin obtained by oxidation of pinB-Bpin. The structures of the C-6 (benzotriazol-1-yl)purine nucleosides have been unequivocally established via Pd-mediated C-N bond formation between bromo purine nucleosides and 1H-benzotriazole. Finally, short and extremely simple synthesis of 1,N6-ethano- and 1,N6-propano-2'-deoxyadenosine are reported in order to demonstrate the synthetic versatility of the O6-(benzotriazol-1-yl)inosine nucleoside derivatives for the assembly of relatively complex compounds.

Solid Phase Synthesis of Nucleobase and Ribose Modified Inosine Nucleoside Analogues

The synthesis and the use of new N-1-dinitrophenyl-inosine based solid support is reported. The support, which binds the nucleoside by a 5 ′-O-monomethoxytrityl function, reacting with N-nucleophiles allowed the synthesis of a small library of N-1 alkylated inosine and AICAR derivatives. Moreover, the obtained supports, after the cleavage of the 2 ′ -3 ′ ribose bond, furnished a set of new N-1 alkylated-2 ′ -3 ′ -secoinosine derivatives in high yields.

Synthesis of 2′,3′-Dideoxyinosine via Radical Deoxygenation

A synthetic method for 2 ′,3 ′-dideoxyinosine (ddI) from inosine was established via radical deoxygenation of N1,5 ′-O-diprotected-2 ′,3 ′-bis-S-methyl dithiocarbonate of inosine derivatives. The radical deoxygenation proceeded smoothly to give the desired dideoxy compounds in good yields using 1-ethylpiperidinium hypophosphite and triethylborane. Benzyl or p-methoxybenzyl protection of inosine at the N1, 5 ′-O-positions were effective for the ddI synthesis.

Microwave‐Assisted Selective 5′‐O‐Trityl Protection of Inosine Derivatives.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.