Synthesis and General Biological Activity of a Small Adenosine-5′-(Carboxamide and Sulfanilamide) Library

A small library of fifty-five adenosine peptide analogs was synthesized, under the Pilot Scale Library (PSL) Program of the NIH Roadmap initiative, from 2′,3′-O-isopropylideneadenosine-5′-carboxylic acid 2. The coupling of amine or sulfanilamide reactants to the free 5′-carboxylic acid moiety of 2, in automated solution-phase fashion, led after acid-mediated hydrolysis to target compounds 3–57 in good yields and high purity. No marked anticancer or antimalarial activity was noted on preliminary cellular testing. Initial screening through the MLPCN program, however, indicates that these analogs may show diverse and interesting biological activities.     

Synthesis of 2′-Deuterio Tubercidin and Adenosine and the Corresponding Arabino Analogs

Abstract

The 2′-deuterio arabino analogs of tubercidin and adenosine have been prepared by Swern oxidation of the 3′,5′-TPDS derivatives of tubercidin and adenosine and reduction with NaBD_4. Subsequent inversion of stereochemistry at C-2′ yielded [2′-²H]tubercidin and [2′-²H]adenosine with 98% deuterium incorporation.     

The First Synthesis of 4′-Branched 5′-Deoxycarbocyclic 9-Deazaadenosine and Phosphonic Acids as Antiviral Agents

The first synthetic route to 4′-trifluoromethylated 5′-deoxycarbocyclic-9-deazaadenosine analog and its phosphonic acid derivatives was described from α-trifluoromethyl-α,β-unsaturated ester. The C–C bond connection between cyclopentane and base moiety was accomplished using Knoevenagel type condensation from ketone derivative 11. Synthesized nucleoside and phosphonic acid analogs were tested for anti-HIV activity as well as cytotoxicity.     

Synthesis and Antiretroviral Activity of Novel 4′-Fluoro-5′-Deoxyphosphonic Acid Carbocyclic Nucleoside Analogs

Novel 5′-deoxycarbocyclic purine phosphonic acid analogs with the 4′-electropositive moiety, fluorine were designed, and synthesized from glyceraldehyde. The cyclopentenol intermediate, 9, was successfully synthesized by the ring-closing metathesis of divinyl 8. The condensation reaction of cyclopentanol 15 with purine bases under Mitsunobu conditions successfully afforded the desired phosphonate analogs. The synthesized nucleoside phosphonic acid analogs, 19, 22, 26, and 29, were subjected to antiviral screening against human immunodeficiency virus (HIV)-1. Guanine phosphonic acid analog 29 showed significant anti-HIV activity (EC₅₀ = 10.3 μM).     

INTRACELLULAR Ca2+-MOBILIZING ADENINE NUCLEOTIDES. SYNTHESIS AND BIOLOGICAL ACTIVITY OF CYCLIC ADP-CARBOCYCLIC-RIBOSE AND C-GLYCOSIDIC ANALOG OF ADENOPHOSTIN A

We designed novel Ca²⁺-mobilizing purine nucleotides, cyclic ADP-carbocycl-icribose 4, and its inosine congener 5, and C-glycosidic adenophostin A 6. In the synthesis of cADPR analogs, the intramolecular condensation to form the pyrophosphate linkage should be the key step. We developed an efficient method for forming such an intramolecular pyrophosphate linkage by the activation of the phenylthiophosphate group with I₂ or AgNO₃. Using this method, we achieved to synthesize the target compounds 4 and 5. The synthesis of C-glycosidic analog 6 of adenophostin A was achieved using a temporary silicon-tethered radical coupling reaction for constructing (3′α, 1″α)-C-glycosi-dic structure as the key step.     

Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Synthesis and Physiological Activity of Methyl 6′6′-Didemethyl Abscisate and New Chiral Analogs

Methyl 6′, 6′-didemethyl abscisate (5) was synthesized and assayed to elucidate the physiological activity of methyl substituents on the cyclohexene ring of abscisic acid (ABA). During this study two new chiral stereoisomeric analogs 6 and 7 were synthesized from l-and d-carvone. The rice seedling assay and germination assay of garden radish showed that 6′-methyl groups of ABA were not important in biological activity and that 5′-isopropenyl analogs 6 and 7 were inactive.     

A Convenient Synthesis of Acyclic Adenosines with an Unsaturated Side Chain by Modification of 9-(2,3-O-Isopropylidene-D-Ribityl)Adenine

Abstract

In expectation of discovering their antiviral activity, acyclic adenosine derivatives 7, 11, 12, and 16 were designed as analogs of neplanocin A (NPA) and L-eritadenine which are strong inhibitors of S-adenosyl-L-homocysteine hydrolase. The 1′,5′-seco-analog of 4′-deoxymethyl-NPA (DHCA) 7 was synthesized by dideoxygenation of 9-(2,3-O-isopropylidene-D-ribityl)adenine (2). Acyclic DHCA analogs 11 and 16 were obtained by Wittig reaction of the aldehyde 3 with Ph₃P=CHCO₂Et and Ph₃P=CHCN, respectively. Hydrolysis of the ester 11 afforded a vinylog of L-eritadenine 12. The synthesized acyclic nucleosides 7, 10, and 11 were evaluated for antiviral activity, however, none of them showed any significant antiviral activity.     

Synthesis and Anti-HIV Activity of Novel 4′-Trifluoromethylated 5′-Deoxycarbocyclic Nucleoside Phosphonic Acids

Efficient synthetic route to novel 4′-trifluoromethylated 5′-deoxycarbocyclic nucleoside phosphonic acids was described from α-trifluoromethyl-α,β-unsaturated ester. Coupling of purine nucleosidic bases with cyclopentanol using a Mitsunobu reaction gave the nucleoside intermediates which were further phosphonated and hydrolyzed to reach desired nucleoside analogs. Synthesized nucleoside analogs were tested for anti-HIV activity as well as cytotoxicity. Adenine analog 22 shows significant anti-HIV activity (EC₅₀ = 8.3 μM) up to 100 μM.     

7‐Deaza‐2′‐Deoxyadenosine‐5′‐Triphosphate as an Alternative Nucleotide for the Pyrosequencing Technology

A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7‐deaza‐2′‐deoxyadenosine‐5′‐triphosphate (c⁷dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2′‐deoxyadenosine‐5′‐O‐(1‐thiotriphosphate) (dATPαS). The inhibitory effect dATPαS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c⁷dATP for the dATPαS. Both analogs show high stability after long time storage at + 8°C. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.     

Transition Metal Complexes of 5-Amino-1-β-D-Ribofuranosylimidazole-4-Carboxylic Acid 5′-Phosphate,an Intermediate in the de novo Biosynthesis of Purine Nucleotides: Synthesis and Effects on Enzyme Activity

Abstract

Transition metal complexes [Cu(II), Co(I1) and Ni(II)] of 5-amino-l-β-D-ribofuranosylimidazole-4-carboxylic acid have been prepared and shown to form a series of stoichiometry ML₂, nM(OH₂) (n = 0,1,2) and structures have been assigned. Analogous complexes of 5-amino-l-β-D-ribofuranosylimidazole-4-carboxy1ic acid 5′-phosphate (CAIR), a central intermediate in the de novo pathway to purine nucleotides, produced in aqueous solution have been found to affect the activity of the enzyme AIR- carboxylase (E.C. 4.1.1.21).     

Partial Protection of Carbohydrate Derivatives. Part 24.1 Synthesis of Adenylyl-(2′–5′)-Adenylyl-(2′–5′)-Adenosine Using a 3′-O-(Tetrahydropyran-2-YL) Adenosine Derivative

Abstract

The synthesis of the title compound was performed using a 3′-O-(tetrahydropyran-2-yl) adenosine derivative as the starting material, i.e., a coupling reaction of triethylammonium N ⁶-benzoyl-5′-O-dimethoxytrityl-3′-O-(tetrahydropyran-2-yl) adenosine 2′-(4-chlorophenyl)phosphate with N ⁶-benzoyl-2′,3′-di-O-benzoyladenosine, followed by a sequence of reactions, O-dedimethoxytritylation, a coupling reaction with the former triethylammonium salt, and complete deblocking of the resultant 2′, 5′-triadenylic acid derivative.     

New Neplanocin Analogues. V. A Potent Adenosylhomocysteine Hydrolase Inhibitor Lacking Antiviral Activity. Synthesis And Antiviral Activity Of 6″-Carboxylic Acid Derivatives Of Neplanocin A 1

Abstract

The 6′-carboxylic acid derivative of neplanocin A 3 was synthesized from NPA, and was converted to the corresponding methyl ester 4 and amides 5 and 6. These were evaluated for their anti-RNA-virus activities. Of the derivatives synthesized, only 5 was active against RNA viruses within the concentration range of 0.14-4.88 μg/mL. Compounds 3 and 5 showed a potent inhibitory effect on S-adenosylhomocysteine (AdoHcy) hydrolase from rabbit erythrocytes. Although a close correlation between the inhibitory effect of adenosine analogues on AdoHcy hydrolase and their antiviral potency has been demonstrated, 3 did not show any anti-RNA-virus activities.

     

INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE WITH FLUORINATED ANALOGS OF 2′- AND 3′-DEOXY-5′-METHYLTHIOADENOSINE

Fluorinated analogs of 2′- and 3′-deoxy-5′-methylthioadenosine 1–4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Design and Synthesis of Dually Branched 5′-Norcarbocyclic Adenosine Phosphonodiester Analogue as a New Anti-HIV Prodrug

A novel 3′,4′-dimethyl-5′-norcarbocyclic adenosine phosphonic acid was prepared using acyclic stereoselective route from 4-hydroxybutan-2-one (4). To improve the cellular permeability and enhance the anti-HIV activity of this phosphonic acid, a (bis)SATE phosphonodiester nucleoside prodrug (20) was prepared and its chemical stability was evaluated. The newly synthesized bis(SATE) analogue (20) and its parent nucleoside phosphonic acid (18) were assayed for anti-HIV activity using an in vitro assay system in a CEM cell line.     

Synthesis of N,N-Dialkylaniline-2′-deoxyuridine Conjugates for DNA-Mediated Electron Transfer Studies

Abstract

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2′-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2′-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O⁴-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination,callus and shoot formation

A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain 1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998–42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5- and 3-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco (Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5- and 3-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy-l-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.Abbreviations A2C: l-Azetidine-2-carboxylic acid - A3C: Azetidine-3-carboxylic acid - Hyp: 4-Hydroxy-l-proline - hpt: Hygromycin phosphotransferase II - NPTII: Neomycin phosphotransferase II Communicated by H. Wang     

SYNTHESIS OF NOVEL 8-SUBSTITUTED CARBOCYCLIC ANALOGS OF 2′,3′-DIDEOXYADENOSINE WITH ACTIVITY AGAINST HEPATITIS B VIRUS

ABSTRACT

Synthesis and antiviral activity of several new 8-substituted carbocyclic analogs of D-2′,3′-dideoxyadenosine are described. The new 8-substituted analogs were synthesized via lithiation of carbocyclic 2′,3′-dideoxy adenosine followed by quenching with electrophiles. This methodology allows for a divergent synthesis of a variety of 8-substituted analogs from carbocyclic 2′,3′-dideoxyadenosine in high yields. 8-Methyl and 8-halogenated carbocyclic 2′,3′-dideoxyadenosine analogs showed 6–25 fold more activity against hepatitis B virus than the unsubstituted carbocyclic D-2′,3′-dideoxyadenosine.     

Synthesis and Incorporation of 5″-Amino- and 5′-Mercapto-5′-Deoxy-2′-O-Methyl Nucleosides Into Hammerhead Ribozymes

Abstract

Novel 5′-amino-5′-deoxy-2′-O-methyl uridine, guanosine and adenosine 3′-O-phosphoramidites 5, 11, and 20, as well as protected 5′-mercapto-5′-deoxy-2′-O-methyl uridine 3′-O-phosphoramidite 23 were synthesized from 2′-O-methyl nucleosides. These analogs were incorporated at the 5′-ends of hammerhead ribozymes to evaluate achiral bridging 5′-N- phosphoramidates and 5′-S-phosphorothioates as alternatives for non- bridging phosphorothioates commonly used for end stabilization against nucleases. Oligonucleotide synthesis and deprotection conditions were optimized for better yields of these modified ribozymes.