Formation and reactivity of 2,4-ditriazolyl pyrimidine C-nucleoside derived from pseudouridine.

Thymine and 2',3',5'-tri-O-acetyl-psi-uridine (1) was converted into the corresponding 2,4-ditriazolyl derivatives 5 and 2, respectively. Of these two substituents, the C4-triazolyl group was found to be quite susceptible to nucleophilic substitution while the other triazolyl is resistant.

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.

Novel mycophenolic adenine bis(phosphonate)s as potential immunosuppressants.

Mycophenolic acid (MPA) is the most potent and specific inhibitor of inosine monophosphate dehydrogenase (IMPDH). This compound was reported to bind the NAD site of IMPDH and mimic the binding of nicotinamide moiety of nicotinamide adenine dicnucleotide. We linked MPA derivatives with the adenine moiety of NAD through a methylenebis(phonphonate) birdge to form novel mycophenolic adenine dinucleotides (MADs) which resemble well the intact natural cofactor. The MAD analogues differ by the length of the side chain (linker) between the aromatic ring of mycophenolic derivative and the beta-phosphorus atom of the adenosine bis(phosphonate) moiety. Regardless of the linker size, MADs were found to be potent inhibitors of human IMPDH type I and type II with Ki's = 0.25-0.52 microM, an order of magnitude less potent than MPA itself (Ki = 0.01-0.04 microM). The growth of K562 cells was inhibited by MPA (IC50 = 0.03 microM) and the MAD analogues (IC50 = 0.01-1.15 microM) with a similar potency. Accordingly, a suppression of alloantigen- induced proliferation of human lymphocytes by the MAD analogues at concentration of 10-20 microM was equally effective as that observed for MPA. In contrast to MPA, MAD analogues were found to be resistant to glucuronidation in vitro. Since therapeutic potential of MPA is limited by its undesirable glucuronidation, the glucuronidation- resistant MAD analogues may be superior immunosuppressants if they are not glucuronidated in vivo.

Synthesis of a novel C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one (2'-deoxy-9-deazaguanosine).

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2'-deoxyguanosine) was achieved starting from 2-amino-6-methyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)-D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by 1H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.

Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM). Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the >10(6)-fold catalytic specificity of UDG. Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism. The first step (E + S left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM). The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 1200 s-1). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.

Targeted gene knockout mediated by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Substrates of DNA polymerases with planar conformation of sugar: model of substrate transition state?

Several years ago, we published an hypothesis concerning conformation of the glycone moiety of different substrates in active centers of several DNA metabolizing enzymes (Nucleosides & Nucleotides 1993, 12, 649-670). This hypothesis prompted us to further study the subtle conformational changes on substrates of DNA polymerases. Data collected in our, as well as other laboratories, have been analyzed, and models of active centers of different DNA polymerases are discussed below. Based on the model of substrate requirements, we now can divide DNA polymerases into two distinguished classes.

Aliphatic and alicyclic diols induce melanogenesis in cultured cells and guinea pig skin.

We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of "supranuclear caps," as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol > 3,3-dimethyl-1,2-butanediol > cis-1,2-cyclopentanediol > 2,3-dimethyl-2,3-butanediol > 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.

Synthesis of a methylenebis(phosphonate) analogue of mycophenolic adenine dinucleotide: a glucuronidation-resistant MAD analogue of NAD.

Mycophenolic alcohol (MPAlc), obtained by reduction of the carboxylic group of mycophenolic acid (MPA), was coupled with 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (4) in the presence of diisopropylcarbodiimide (DIC) to give P1-(2',3'-O-isopropylideneadenosin-5'-yl)-P2-(mycophenolic alcohol-6'-yl)methylenebis(phosphonate) (8) in 32% yield. Deisopropy-lidenation of 8 with CF3COOH/H2O afforded the methylenebis(phosphonate) analogue 3 of mycophenolic adenine dinucleotide (MAD). Compound 3, beta-methylene-MAD, was found to be a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II (Ki = 0.3 microM) as well as an inhibitor of growth of K562 cells (IC50 = 1.5 microM). In contrast to MPA and mycophenolic alcohol, beta-methylene-MAD was not converted into the glucuronide when incubated with uridine 5'-diphosphoglucuronyltransferase.

Synthesis of nonhydrolyzable analogues of thiazole-4-carboxamide and benzamide adenine dinucleotide containing fluorine atom at the C2' of adenine nucleoside: induction of K562 differentiation and inosine monophosphate dehydrogenase inhibitory activity.

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

The practical synthesis of a methylenebisphosphonate analogue of benzamide adenine dinucleotide: inhibition of human inosine monophosphate dehydrogenase (type I and II).

beta-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD), was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase (IMPDH). Treatment of 2',3'-O-isopropylideneadenosine 5'-methylenebisphosphonate (15) with DCC afforded P1,P4-bis(2',3'-O-isopropylideneadenosine) 5'-P1,P2:P3,P4-dimethylenetetrakisphosphonate (17). This compound was further converted with DCC to an active intermediate 18 which upon reaction with 3-(2',3'-O-isopropylidene-beta-D-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired beta-methylene-BAD (8) in 95% yield. In a similar manner, treatment of 18 with 2',3'-O-isopropylidenetiazofurin (21) followed by hydrolysis and deprotection afforded beta-methylene-TAD (5) in 91% yield. Compound 8 (IC50 = 0.665 microM) was found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC50 = 0.107 microM) and was almost equally potent against IMPDH type I and type II. Although TAD and beta-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki = 333 microM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki = 6.3 microM).

Cyclopent[a]anthraquinones as DNA intercalating agents with covalent bond formation potential: synthesis and biological activity.

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently. Structure-activity relationships were studied. Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl] cyclopent[alpha]anthracene-6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (i.p., QD x 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound 4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to intercalate into DNA double strands and is also a topoisomerase II inhibitor.

Chemical synthesis of benzamide adenine dinucleotide: inhibition of inosine monophosphate dehydrogenase (types I and II).

Treatment of 3-(2,3-O-isopropylidene-beta-D-ribofuranosyl)benzamide (6) with POCl3 in (EtO)3-PO afforded only little phosphorylation product (8, 5%), but the major product was 5'-chlorobenzamide riboside (7, 85%). Reaction of 6 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite followed by 2-cyanoethanol/tetrazole treatment and oxidation with tert-butyl peroxide gave a 1:1 mixture of the desired 5'-O-bis(2-cyanoethyl) phosphate 9 and the chloro derivative 7. This mixture was treated with methanolic ammonia and partitioned between CHCl3 and water. The 2',3'-O-isopropylidenebenzamide mononucleotide (8) was obtained in 21.2% overall yield from the aqueous layer. Compound 8 was then converted into the corresponding imidazolide 11b which, upon coupling with 2',3'-O-acetonide of AMP, afforded the acetonide of benzamide adenine dinucleotide (15) in 94% yield together with small amounts of symmetrical pyrophosphates P1,P2-bis(2',3'-O-isopropylideneadenosin-5'-yl)pyrophosphate (13, 3%) and P1,P2-bis(2',3'-O-isopropylidene-3-(carbamoylphenyl)-5'-ribosyl)py rophosphate (14, 2%). Deprotection of 15 with Dowex 50/H+ in water afforded the desired benzamide adenine dinucleotide (BAD) in 93% yield. BAD inhibits inosine monophosphate dehydrogenase type I (IC50 = 0.78 microM) and type II (IC50 = 0.88 microM) with same degree of potency.

Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

NAD-analogues as potential anticancer agents: conformational restrictions as basis for selectivity.

Cofactor type inhibitors (NAD-analogues) of IMP-dehydrogenase (IMPDH) were synthesized and their application as potential anticancer agents are discussed. C-nucleoside isosteres of NAD, C-NAD and C-PAD, showed an effective competitive inhibition of IMPDH, C-NAD but not C-PAD caused extremely potent inhibition of alcohol dehydrogenase. We also synthesized compounds in which nicotinamide riboside was replaced with tiazofurin (TAD-analogues) and the 2' and 3'-positions of adenosine part were fluorinated. The ribose ring of 2'-deoxy-2'-fluoroadenosine is in the C3'-endo conformation whereas 3'-deoxy-3'-fluoroadenosine favors the C2'-endo sugar pucker. These derivatives are good inhibitors of IMPDH type II, the isoenzyme dominant in neoplastic cells. In contrast, all these analogues showed rather week inhibitory activity against alcohol dehydrogenase. Nicotinamide riboside derivatives in which the base and the sugar are linked through an oxygen or a methylene bridge were synthesized. NAD-analogues containing such conformationally restricted nicotinamide nucleoside moiety (syn or anti) are expected to be selective inhibitors of B-specific (IMPDH) or A-specific dehydrogenases, respectively.

9-substituted acridine derivatives with long half-life and potent antitumor activity: synthesis and structure-activity relationships.

A series of DNA-intercalating 9-anilinoacridines, namely 9-phenoxyacridines, 9-(phenylthio)acridines, and 9-(3',5'-disubstituted anilino)acridines, were synthesized as potential antitumor agents with inhibitory effects on DNA topoisomerase II. Unlike amsacrine (m-AMSA), these agents were designed to avoid the oxidative metabolic pathway. These acridine derivatives were, therefore, expected to have long half-life in plasma. Both 9-phenoxyacridines and 9-(phenylthio)acridines were found to have moderate cytotoxicity against mouse leukemia L1210 and human leukemic HL-60 cell growth in culture. Among 9-(3',5'-disubstituted anilino)acridines, 3-(9-acridinylamino)-5-(hydroxymethyl)aniline (AHMA) was found to be a potent topoisomerase II inhibitor and exhibited significant antitumor efficacy both in vitro and in vivo. Chemotherapy of solid-tumor-bearing mice with 10, 10, and 5 mg/kg (QD x 4, ip) AHMA, VP-16, and m-AMSA, respectively, resulted in more tumor volume reduction by AHMA than by VP-16 or m-AMSA for E0771 mammary adenocarcinoma and B-16 melanoma. For Lewis lung carcinoma, AHMA was as potent as VP-16 but more active than m-AMSA. Structure-activity relationships of AHMA derivatives are discussed.

Synthesis of 2'-fluoro-5-[11C]-methyl-1-beta-D-arabinofuranosyluracil ([11C]-FMAU): a potential nucleoside analog for in vivo study of cellular proliferation with PET.

Rapid in vivo catabolism limits the use of currently available radiotracers used in tumor proliferation studies with PET. This is manifested by the need to develop complex mathematical models to interpret kinetic and metabolite data obtained from imaging studies with agents such as carbon-11 labeled thymidine. A potential carbon-11 labeled radiotracer for cellular proliferation, 2'-fluoro-5-([11C]-methyl)-1-beta-D-arabinofuranosyluracil (FMAU), has been prepared using a previously described method for preparation of [11C]methyl-thymidine where selective alkylation of a pyrimidyl dianion is accomplished with [11C]methyl iodide at the 5-position of the pyrimidine ring. FMAU shares many in vivo characteristics of thymidine, including cellular transport, phosphorylation by mammalian kinase, and incorporation into DNA. Most importantly, in vivo catabolism of FMAU is limited, potentially yielding simplified kinetic models for determination of cellular proliferation with positron emission tomography.

Crystallographic studies of isosteric NAD analogues bound to alcohol dehydrogenase: specificity and substrate binding in two ternary complexes.

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric C-glycosidic analogue of NAD(H) containing a neutral pyridine ring. CPAD (5-beta-D-ribofuranosylpicolinamide adenine dinucleotide) is a closely related pyridine-containing analogue with the pyridine nitrogen on the opposite side of the ring. CNAD is a potent and specific inhibitor of horse liver alcohol dehydrogenase (LADH), binding with a dissociation constant in the nanomolar range. CPAD binds LADH with an affinity comparable to that of NAD. Crystal structures of CNAD and CPAD bound to LADH are presented at 2.4 and 2.7 A, respectively. The two complexes are isomorphous, crystallizing in the triclinic system with cell dimensions different from those seen in previous ternary LADH complexes. Structures were solved using the molecular replacement method and refined to crystallographic R values of 18% (CNAD) and 17% (CPAD). Both inhibitors bind to the "closed" form of LADH in the normal cofactor-binding cleft. The conformation of LADH-bound CPAD closely mimics that of LADH-bound NAD(H). The data suggest that alcohol substrate binds directly to the catalytic zinc atom. In the CNAD complex, the pyridine nitrogen replaces alcohol as the fourth coordination ligand to the active site zinc atom, while all other polar interactions remain the same as those of bound NAD(H). The zinc-nitrogen ligand explains the high affinity of CNAD for LADH.

Synthesis and biological properties of 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil.

A novel 5-o-carboranyl-containing nucleoside, 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (6, CFAU), was synthesized as a potential intracellular neutron capture agent. This compound was prepared in five steps starting from 5-iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (1). The desired carboranyl derivative was obtained by addition of decaborane [as the bis(propionitrile) adduct] to the protected acetylenic nucleoside precursor followed by debenzoylation. The synthesis of CFAU was also performed by glycosylation of the suitably protected 5-o-carboranyluracil with the appropriate 2-fluoroarabinosyl derivative. This compound was evaluated for its cytotoxicity in human lymphocytes, monkey cells, and rat and human gliomas cells, as well as for antiviral activity against human immunodeficiency virus and herpes simplex virus type 1. Its biological activity was compared to 5-o-carboranyl-1-(2-deoxyribofuranosyl)uracil in these cell culture systems, human bone marrow cells, and mice. The results obtained to date suggest that CFAU has suitable characteristics as a sensitizer for boron neutron capture therapy.

CNAD: a potent and specific inhibitor of alcohol dehydrogenase.

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric and isomeric analogue of NAD, in which the nicotinamide ring is linked to the sugar via a C-glycosyl (C5-C1') bond. CNAD acts as a general dehydrogenase inhibitor but shows unusual specificity and affinity for liver alcohol dehydrogenase (ADH, EC 1.1.1.1). The pattern of inhibition is congruent to 4 nM, with NAD as the variable substrate. These values are 3-5 orders of magnitude smaller than those obtained for CNAD in other dehydrogenases and are comparable to values observed for the tightest binding ADH inhibitors known. The specificity and affinity of CNAD for ADH are likely due to coordination of the zinc cation at the ADH catalytic site by the CNAD pyridine nitrogen. This is supported by kinetic and computational studies of ADH-CNAD complexes. These results are compared with those for a related analogue, CPAD. In this analogue, displacement of the pyridine nitrogen to the opposite side of the ring removes the specificity for ADH.