Substrate For Adenosine Deaminase Research Articles

Emerald Jewel Wasp Venom Adenosine Deaminase Antagonizes Purinergic Signaling in the Cockroach Brain

Ampulex compressa injects venom directly into the cerebral ganglia of the cockroach (Periplaneta americana) to induce a reversable, but long‐term compliant state of hypokinesia, marked by an increase in escape threshold and a decrease in spontaneous walking. The venom is not paralytic as stung cockroaches can still stand, groom, eat and drink, and can even run if aggressively provoked. The venom is also not necrotic or cytotoxic, and despite its long‐lasting duration the venom’s effects are reversible, as the escape response returns to normal after several days. Previous work involved generation of a comprehensive venom apparatus transcriptome and venom proteome or “venome” and described several interesting signaling pathways that appear to be targeted by venom components. This includes adenosine deaminase (ADA), which may act to antagonize purinergic signaling in the brain.The substrates of venom ADA, adenosine (Ado) and adenosine monophosphate (AMP), are also the activating ligands of the cockroach adenosine receptor (AdoR). We hypothesize that the presence of ADA at the site of injection can reduce cell damage‐induced signaling by clearing Ado and AMP released from compromised cells. We have cloned, expressed, and characterized both venom ADA and cockroach brain AdoR and show that the presence of venom ADA can attenuate Ado induced calcium signaling in vitro. Antagonizing ADA or AdoR in vivo did not alter locomotory behavior. Thus, the action of ADA in the venom may be more neuroprotective than inhibitory of specific behaviors as is the case with other venom components.

Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination.

We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

A first-in-human study of, NUC-7738, a 3'dA phosphoramidate, in patients with advanced solid tumors or lymphoma (NuTide 701).

TPS3147 Background: Nucleoside analogs form the backbone therapy for both hematological and solid malignancies. However, their clinical effectiveness is severely limited by key cellular resistance mechanisms linked to increased breakdown, impaired activation and transport. NUC-7738 is a phosphoramidate transformation of cordycepin (3’-deoxyadenosine; 3’-dA), a derivative of adenosine that was first isolated from Cordyceps sinensis. The cytotoxic effect of 3’-dA is largely attributed to intracellular generation of the triphosphate metabolite, 3’-dATP, terminating DNA and RNA synthesis. Although 3’-dA has shown potent anti-tumor activity in non-clinical studies, it has not been successful in clinical studies mainly because of rapid enzymatic degradation by adenosine deaminase. NUC-7738 is not a substrate for adenosine deaminase and has been designed to bypass the key resistance pathways which have limited the clinical effectiveness of cordycepin. Methods: NuTide:701 is a two-part, first-in-human Phase I study in patients with advanced solid tumors and lymphoma who have exhausted all standard treatment options. The primary objective is to determine the RP2D and schedule of NUC-7738. Secondary objectives include safety, PK/PD and anti-tumor activity. Part 1, in patients with advanced solid tumors, will establish the RP2D and dose administration schedule of NUC-7738 for Part 2. Part 2 will further evaluate the selected RP2D and designated dosing schedule in an expansion cohort of patients with advanced solid tumors or lymphoma. The study initiated in Q1 2019. Clinical trial information: NCT03829254.

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

FRI0472 The level of adenosine deaminase activity in synovial fluid in patients with rheumatoid arthritis and osteoarthritis

BackgroundDuring last years, the great attention is paid to the identification of biomarkers which can help in differentiating the type of arthritis. The recent studies have shown Adenosine deaminase (ADA)...

ADA-SCID with ‘WAZA-ARI’ mutations that synergistically abolished ADA protein stability

ADA-SCID with ‘WAZA-ARI’ mutations that synergistically abolished ADA protein stability

Homologous Recombination Repair of DNA Strand Breaks Induced by the Nucleoside Analogue CNDAG.

AbstractAbstract 1189We hypothesize that the novel deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl) guanine] may share the common action mechanism with its cytosine congener CNDAC [2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine], a prodrug of which, sapacitabine, is undergoing clinical trials in myeloid leukemias. CNDAC induces single strand breaks following incorporation into DNA. Subsequent processing or DNA replication across the unrepaired nicks would generate double strand breaks (DSBs) [1]. Because cytosine and guanine nucleoside congeners have remarkably different clinical activities, e.g., cytarabine (acute myelogenous leukemia) and nelarabine (T-cell malignancies), it will be useful to pursue investigations to fully characterize the metabolism and actions of CNDAG. This study was aimed at defining cellular response and damage repair mechanisms for two CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each prodrug is a substrate for adenosine deaminase (ADA), the action of which generates CNDAG. First, growth inhibition by both CNDAG prodrugs was dependent upon both concentration and time of exposure; the proliferation of T-cell malignant lines (CCRF-CEM and Jurkat) was suppressed more to B-cell lines (Raji and IM-9). This may be attributed to relatively low activity of deoxycytidine kinase in the latter cell lines. Second, p53 knocked-out and parental HCT116 cells were equally sensitive to CNDAG 6-NH2 in a clonogenic assay, indicating that cytotoxicity of CNDAG is independent of p53 status. Third, similar to CNDAC, CNDAG prodrugs activated repair proteins in multiple DNA damage response pathways, as revealed by immunoblotting. 24-hr incubation of CCRF-CEM cells with 50 microM either prodrug increased the phosphorylation of Ser-1981 on ATM, Ser-345 on Chk1, Thr-68 on Chk2, Ser-966 on SMC1, Ser-343 on Nbs1 and g-H2AX. In contrast, there was no increase in phosphorylation of two other sensor kinases, DNA-PKcs (Ser-2056) which participates in repair of double strand breaks by non-homologous end-joining, and ATR (Ser-428) which senses stalled DNA replication forks. Fourth, we investigated the role of components of homologous recombination (HR) in CNDAG-induced DNA damage repair. The clonogenic survival of human fibroblasts deficient in ATM or those transfected with an empty vector were approximately 20- to 30-fold more sensitive to CNDAG prodrugs than cells complemented with full-length ATM cDNA. Chinese hamster cells deficient in Rad51D or either of the two Rad51-interacting proteins, Xrcc3 and Brca2, conferred greater than 30-fold sensitivity to CNDAG prodrugs relative to respective wild type lines. Similar sensitization was also observed with CNDAC. In contrast, cells lacking HR function were not more sensitive to ara-C or ara-G compared to their parental and complemented cells, indicating HR is a unique repair mechanism for 2'-C-cyano-2'-deoxy-nucleoside analogues. Finally, a cytogenetic approach was used to analyze sister chromatid exchange (SCE, a hallmark for HR) formation in metaphase cells exposed to 2 microM CNDAG 6-NH2. The frequencies of SCEs in AA8 cells incubated with CNDAG for two cell cycles (mean 14.2 per metaphase) were 2-fold of those exposed for one cell cycle (mean 7.4 per metaphase, n>20, p<0.001), the latter greater than control (mean 6 per metaphase, p<0.05). Together these results demonstrate that DNA damage caused by CNDAG activates ATM-dependent signaling pathways and is repaired through homologous recombination. Thus, this is a class effect caused by 2'-C-cyano-2'-deoxy-nucleoside analogues. Our study suggests that despite relatively less potency, CNDAG might have distinct clinical activity from that of CNDAC. [1] Liu X, et al. Blood, Blood. 2010 May 17, Epub ahead of print, PMID: 20479284. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

ChemInform Abstract: Stereoselective Synthesis of Indazole 2′-Deoxy-β-D- ribonucleosides: Glycosylation of the Nucleobase Anion.

The glycosylation of indazolyl anions derived from 4a, b with 2-deoxy-3,5-bis-O-(4-methylbenzoyl)-α-D-erythro-pentofuranosyl chloride (5) is described. The reaction was Stereoselective – exclusive β-D-anomer formation – but regioisomeric N1- and N2-(2′-deoxy-β-D-ribofuranosides) (i.e.6a and 7a, resp., and 6b and 7b, resp.) were formed in about equal amounts. They were deprotected to yield 8a, b and 9a, b. Compound 1, related to 2′-deoxyadenosine (3), and its regioisomer 2 were obtained from 8b and 9b, respectively, by catalytic hydrogenation. The anomeric configuration as well as the position of glycosylation were determined by 1D NOE-difference spectroscopy. The first protonation site of 1 and 2 was found to be the NH2 group. The N-glycosylic bond of 1H-indazole N1-(2′-deoxyribofuranosides) is more stable than that of the parent purine nucleosides. Compound 1 is no substrate for adenosine deaminase.

ChemInform Abstract: Synthesis and Biological Evaluation of 4-Purinylpyrrolidine Nucleosides.

The synthesis of several novel carbocyclic purine nucleosides that incorporate a nitrogen in place of carbon 3 of the cyclopentyl moiety are described. These analogues are all derived from the key stereochemically defined intermediate N-(tert-butoxycarbonyl)-O-[(4-methoxyphenyl)diphenylmethyl]-trans- 4- hydroxy-D-prolinol (19), which was accessible in 61.1% overall yield for a five-step sequence starting from cis-4-hydroxy-D-proline. The heterocyclic bases, 6-chloropurine and 2-amino-6-chloropurine, are efficiently introduced onto the pyrrolidine ring via a Mitsunobu-type coupling procedure with triphenylphosphine and diethyl azodicarboxylate. Standard transformations and removal of protecting groups gave the cis-adenine (26), hypoxanthine (27), 2,6-diaminopurine (28), and guanine (29) D-prolinol derivatives. In addition, a related sequence from trans-4-hydroxy-L-proline provided the enantiomeric L-prolinol guanine derivative (36). Lastly, the 6-(dimethylamino)purine analogue, 37, was coupled to N-(benzyl-oxycarbonyl)-p-methoxy-L-phenylalanine to provide, after deprotection, the novel puromycin-like analogue 39. The analogues 26-29, 36, and 39 were all evaluated for antitumor and, except for 39, for antiviral activity. These compounds failed to appreciably inhibit the growth of P388 mouse leukemia cells in vitro at concentrations up to 100 micrograms/mL. In addition, they did not exhibit noticeable activity against the human immunodeficiency virus or herpes simplex virus type 1 at concentrations as high as 100 microM. The adenine analogue, 26, did, however, prove to be a substrate for adenosine deaminase. It possessed an affinity for the enzyme only 50% less than that of adenosine with a Ki = 85 microM.

ChemInform Abstract: Ynamines Derived from Nucleic Acids Bases: Synthesis, Reactivity, and Biological Activity.

Ynamines 1a–d are prepared by alkylation of nucleic acids bases with tetrachloroethylene followed by elimination of halogens from the intermediates 4a–d; reaction of 1a with acetone and cyclohexanone gives carbinols 8a and b and in the case of 8b cyclic ketal 9a is also obtained; compound 1a is a substrate for adenosine deaminase.

ChemInform Abstract: Spiropentane Mimics of Nucleosides: Analogues of 2′-Deoxyadenosine and 2′-Deoxyguanosine. Synthesis of All Stereoisomers, Isomeric Assignment, and Biological Activity.

Synthesis of spirocyclic analogues of 2'-deoxyadenosine and 2'-deoxyguanosine (12a-15a and 12b-15b) is described. Rhodium-catalyzed reaction of ethyl diazoacetate with methylenecyclopropane 19, obtained from 2-bromo-2-bromomethylcyclopropane 17 via debromination (16), reduction (18), and acetylation (19), gave a mixture of all four isomeric spiropentanes 20a-20d. Hydrolysis afforded hydroxy carboxylic acids 21a-21d. Acetylation of separated proximal + medial-syn isomers 21a + 21b and medial anti + distal isomers 21c + 21d furnished acetates 22a + 22b and 22c + 22d. Curtius rearrangement effected by diphenylphosphoryl azide in tert-butyl alcohol performed separately with mixtures 22a + 22b and 22c + 22d led to BOC-amino spiropentanes 23a + 23b and 23c + 23d. After deacetylation all isomers 24a-24d were separated and deprotected to give aminospiropentane hydrochlorides 25a-25d. Free bases were of limited stability. The heterocyclic moieties were introduced into individual isomers 25a-25d via 6-chloropurine derivatives 26a-26d or 30a-30d. Ammonolysis of 26a-26d furnished the adenine isomeric series 12a-15a, whereas guanine derivatives 12b-15b were obtained by hydrolysis of 30a-30d with formic acid. The isomeric assignments followed from IR spectra of BOC-aminospiropentanes 24a-24d and NMR spectra of 12a-15a including NOE and (H,H) COSY. The proximal and medial-syn isomers 12a and 12b were modest inhibitors of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in culture, whereas the medial-anti isomer 12c was a substrate for adenosine deaminase. The distal isomer 15b was an anti-EBV agent. The medial-syn phosphoralaninate 34 was an effective inhibitor of HCMV replication in vitro. It was also active against herpes simplex virus type 1 (HSV-1), varicella zoster virus (VZV), human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), and EBV with a varying degree of cytotoxicity.

(Z)- and (E)-2-(1,2-Dihydroxyethyl)methylenecyclopropane Analogues of 2′-Deoxyadenosine and 2′-Deoxyguanosine. Synthesis of All Stereoisomers, Absolute Configuration, and Antiviral Activity

Chiral Z- and E-stereoisomers of (1,2-dihydroxyethyl)methylenecyclopropane analogues of 2'-deoxyadenosine and 2'-deoxyguanosine were synthesized, and their antiviral activity was investigated. (S)-Methylenecyclopropylcarbinol (16) was converted in seven steps to reagents 26 and 27, which were used for alkylation-elimination of adenine and 2-amino-6-chloropurine to get ultimately analogues 12a, 12b, 13a, 13b, 14a, 14b, 15a, and 15b. The enantiomeric series ent-12a, ent-12b, ent-13a, ent-13b, ent-14a, ent-14b, ent-15a, and ent-15b was obtained by similar procedures starting from (R)-methylenecyclopropylcarbinol (ent-16). The Z-isomer ent-12b was an inhibitor of two strains of human cytomegalovirus (HCMV) with EC(50) of 6.8 and 7.5 microM and of murine cytomegalovirus (MCMV) with EC(50) of 11.3 microM. It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC(50) of 8 microM. The E-isomers ent-15a, ent-13a, and 15b were less effective. All adenine analogues with the exception of the Z-isomers ent-12a and ent-14a were moderate substrates for adenosine deaminase.

Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients

AbstractMutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2′-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4+ T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca2+ flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-κB. Moreover, exposure to 2′-deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A2A adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials.gov as #NCT00598481 and #NCT0059978.

Fluorinated methylenecyclopropane analogues of nucleosides. Synthesis and antiviral activity of ( Z)- and ( E)-9-{[(2-fluoromethyl-2-hydroxymethyl)-cyclopropylidene]methyl}adenine and -guanine

Synthesis and antiviral activity of the title fluoromethylenecyclopropane analogues 15a, 15b, 16a, and 16b is described. Methylenecyclopropane carboxylate was first transformed to 2,2-bis-hydroxymethylmethylenecyclopropane. Selective monoacetylation followed by introduction of fluorine gave 2-acetoxymethyl-2-fluoromethylmethylenecyclopropane as the key intermediate. The synthesis of analogues 15a, 15b, 16a, and 16b then followed alkylation–elimination procedure as described previously for other methylenecyclopropane analogues. The adenine Z-isomer 15a was found to be a potent inhibitor of Epstein–Barr virus (EBV) in vitro with EC 50/CC 50 (μM) 0.5/55.7. Compounds 15b, 16a, and 16b were also active but at higher concentrations, EC 50/CC 50 (μM) 3.2–7.5/53.6–64.1. Analogue 15a inhibited hepatitis C virus by virtue of its cytotoxicity and it moderately inhibited replication of the Towne strain of human cytomegalovirus (HCMV). The E-isomer 16a was a substrate for adenosine deaminase, whereas the Z-isomer 15a was not deaminated.

Methylene-2-ethynylcyclopropanes: synthesis and biological activity of ( Z)- and ( E)-9-{[2-ethynyl-2-(hydroxymethyl)cyclopropylidene]methyl}adenine and -guanine

Synthesis of methylene-2-ethynylcyclopropane analogues of nucleosides 12a, 12b, 13a, and 13b is described. Ethyl methylenecyclopropane carboxylate 14 was hydroxymethylated to give alcohol 15, which was reduced to diol 16. Selective protection with tert-butyldimethylsilyl group gave derivative 17, which was oxidized to aldehyde 18. Wittig reaction with CBr 4 gave dibromoalkene 19. Elimination of both bromine atoms afforded methylene-2-ethynylcyclopropane 20. Bromoselenenylation using N-bromosuccinimide and diphenyldiselenide gave intermediate 21. Alkylation of adenine and 2-amino-6-chloropurine with 21 provided the Z, E-isomeric mixtures 22a and 22c. Oxidation afforded selenoxides 23a and 23c. Mild thermolysis furnished methylenecyclopropanes Z-24a , E-24a , and 24c. Deprotection and separation of Z, E-isomers gave adenine analogues 12a and 13a, and 2-amino-6-chloropurine intermediates 12c and 13c. Hydrolytic dechlorination of 12c and 13c afforded guanine analogues 12b and 13b. Adenine Z-isomer 12a inhibits replication of Epstein-Barr virus through its cytotoxicity. The E-isomer 13a is a substrate for adenosine deaminase.

9-{[3-Fluoro-2-(hydroxymethyl)cyclopropylidene]methyl}adenines and -guanines. Synthesis and Antiviral Activity of All Stereoisomers

All stereoisomers of adenine and guanine methylene-3-fluoromethylenecyclopropane analogues of nucleosides 9a, 9b, 10a, 10b, 11a, 11b, 12a, and 12b were synthesized and their antiviral activities were evaluated. A highly convergent approach permitted the synthesis of all these analogues using a single intermediate 15. Reaction of aldehyde 13 with fluorotrichloromethane and tri-n-butylphosphine gave fluoroalkenes 14a+14b (83:17). Addition of carbene derived from ethyl diazoacetate gave cyclopropane 15 as the major product. Reduction (19), bromination (20), and phenylselenenylation (21), followed by Se oxidation and beta-elimination gave cis-methylenecyclopropane 22. Addition of bromine provided the reagent 23 for alkylation-elimination. Reaction of 23 with adenine led to an isomeric mixture 25a+26a that after deprotection afforded analogues 9a and 10a. The 2-amino-6-chloropurine furnished 25e+26e and after deblocking (9e and 10e) and hydrolysis gave targets 9b and 10b. Intermediate 15 provided, after debenzylation (27), 2-nitrophenylselenenylation (28), reduction (29), benzylation (30), and oxidation-elimination trans-methylenecyclopropane 31. Addition of bromine gave reagent 32. Further transformations followed the sequence outlined for analogues 9a, 9b, 10a, and 10b. Analogue 9b was effective against human cytomegalovirus (HCMV; Towne) with EC50 2.9 microM. The trans-isomer 10b inhibited AD169 strain of HCMV (EC50 15 microM) and the murine virus MCMV (EC50 2.5 microM). Compound 12a was effective against Epstein-Barr virus (EC50<0.03 microM). Analogue 9a inhibited varicella zoster virus (EC50 5.9 microM) and human immunodeficiency virus type 1 (EC50 5.2 microM). Analogues 9a, 10a, and 11a are moderate substrates for adenosine deaminase. The structure-activity relationships will be discussed in context with other methylenecyclopropane analogues.

Oxanosine Is a Substrate of Adenosine Deaminase. Implications for the Quest for a Toxicological Marker for Nitrosation Activity

Oxanosine 3r, 5-amino-3-beta-(d-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazine-7-one, was isolated as a novel nucleoside antibiotic in 1981 from Streptomyces capreolus MG265-CF3. Oxanosine became relevant in toxicology in 1996 with the discovery that it is formed in nitrosative guanosine deamination. As part of studies of the mechanism of oxanosine formation, the synthesis was attempted of [7- 18O]oxanosine by enzymatic 16O/18O-exchange with adenosine deaminase (ADA) in analogy to the synthesis of [6- 18O]guanosine from 2-amino-6-chloropurine. Unexpectedly, it was discovered that the incubation of oxanosine 3r with ADA in sodium phosphate buffer (pH = 7.4) results in 1-beta-(d-ribofuranosyl)-5-ureido-1H-imidazole-4-carboxylic acid 4r. The reaction of the 2'-deoxyribose derivative 3d forms 4d in analogy. The reaction products were separated by preparative RP-HPLC and characterized by LC/MS and MS/MS analyses and UV/vis and NMR spectroscopy, and NMR assignments were corroborated by GIAO and GIAO-PCM calculations. Reaction in H2 18O leads to 18O-incorporation at C7. The hydrolysis of 3 to 4 can be rationalized on the basis of the known mode of action of ADA, and an explanation is provided for ADA's accomplishment of the "usual" substitution at C6 of adenosine (addition to the exocyclic bond) and the "lactone hydrolysis" of oxanosine (addition to the endocyclic double bond). The Michaelis-Menten constant of Km = 1.0 (+/-0.2) mM was measured for oxanosine. Implications are discussed for studies of nitrosative deamination of nucleosides, nucleotides, and oligonucleotides.

(Z)- and (E)-[2-Fluoro-2-(hydroxymethyl)cyclopropylidene]methylpurines and -pyrimidines, a New Class of Methylenecyclopropane Analogues of Nucleosides: Synthesis and Antiviral Activity

The Z- and E-isomers of fluoromethylenecyclopropane analogues 11a-d and 12a-d were synthesized, and their antiviral activities were evaluated. The purine (Z,E)-methylenecyclopropane carboxylates 13 and 24 were selectively fluorinated using lithium diisopropylamide, LiCl, and N-fluorobenzenesulfonimide to give (Z,E)-fluoroesters 22 and 25. Reduction with LiBH(4) or diisobutylaluminum hydride gave after chromatographic separation Z-isomers 11a and 11e and E-isomers 12a and 12e. The O-demethylation of 11e and 12e afforded guanine analogues 11b and 12b. Fluorination of (Z,E)-cytosine and thymine esters 15 and 16 afforded (Z,E)-fluoroesters 26 and 27, which were resolved before the reduction to analogues 11c and 11d and 12c and 12d. Adenine Z-isomer 11a was the most effective against Towne and AD169 strains of human cytomegalovirus (HCMV, EC(50) 3.6 and 6.0 microM, respectively), but it was less effective against murine virus (MCMV, EC(50) 69 microM). Thymine Z-isomer 11d was effective against HSV-1 in BSC-1 cells (ELISA, EC(50) 2.5 microM) but inactive against HSV-1 or HSV-2 in Vero or HFF cells. All of the analogues with the exception of 12d were effective at least in one of the assays against Epstein-Barr virus (EBV) in Daudi or H-1 cells in a micromolar or submicromolar range. Cytosine and thymine Z-isomers 11c and 11d were active against varicella zoster virus (VZV) with EC(50) 0.62 microM. Adenine Z- and E-isomers 11a and 12a were effective against HIV-1 in MT-2 or MT-4 cells with EC(50) 12-22 and 2.3-7.6 microM, respectively, whereas only 12a was effective against hepatitis B virus (HBV) with EC(50) 15 microM. Analogues 11a and 12a were weak substrates for adenosine deaminase.

Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase high performance liquid chromatography: Identification of a novel metabolite of 2-chlorodeoxyadenosine

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5′-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3 h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 μg/ml in the assay. An amount of 8×10 3 cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.

AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus.

5-Aminoimidazole-4-carboxamide riboside (AICA riboside; Acadesine) activates AMP-activated protein kinase (AMPK) in intact cells, and is reported to exert protective effects in the mammalian CNS. In rat cerebrocortical brain slices, AMPK was activated by metabolic stress (ischaemia > hypoxia > aglycaemia) and AICA riboside (0.1-10 mm). Activation of AMPK by AICA riboside was greatly attenuated by inhibitors of equilibrative nucleoside transport. AICA riboside also depressed excitatory synaptic transmission in area CA1 of the rat hippocampus, which was prevented by an adenosine A1 receptor antagonist and reversed by application of adenosine deaminase. However, AICA riboside was neither a substrate for adenosine deaminase nor an agonist at adenosine receptors. We conclude that metabolic stress and AICA riboside both stimulate AMPK activity in mammalian brain, but that AICA riboside has an additional effect, i.e. competition with adenosine for uptake by the nucleoside transporter. This results in an increase in extracellular adenosine and subsequent activation of adenosine receptors. Neuroprotection by AICA riboside could be mediated by this mechanism as well as, or instead of, by AMPK activation. Caution should therefore be exercised in ascribing an effect of AICA riboside to AMPK activation, especially in systems where inhibition of adenosine re-uptake has physiological consequences.