2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract

The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [³²P]Adenylate and [³²P]- and [³H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A₃ core and 2′,5′-3′dA₃ core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[³H]A₃ showed that 0.25% of the ³²P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p₃A₄[³²P]pCp radiobinding assay, 2′,5′-A₃ was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [³H]- and [³²P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although there was no 5′-rephosphorylation as determined by radiobinding assay, the intact cordycepin trimer core was detected by tlc, radiobinding assay, and HPLC.

Furthermore, in two experiments, the concentration of the cordycepin trimer core bound to or taken up by the lymphocytes was three-fold greater than the concentration in the medium. 2′,5′-A₃ and 2′,5′-3′dA₃ cores were both antimitogenic, but did not inhibit protein synthesis.     

Synthesis and Antiviral Activity Assay of Novel (E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine

Abstract

(E)-3′,5′-diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Nucleosides and Nucleotides. 192. Toward the Total Synthesis of Cyclic ADP-Carbocyclic-Ribose. Formation of the Intramolecular Pyrophosphate Linkage by a Conformation-Restriction Strategy in a Syn-form Using a Halogen Substitution at the 8-Position of the Adenine Ring

Abstract

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N ⁶-trichloroacetyl-2′,3′-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5′,5′-diol derivatives 18. When 18 was treated with POCl₃ in PO(OEt)₃, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5′,5′-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.     

Bromine-Induced Selective N-Monodemethylation of N6, N6-Dimethyl-2′,3′-O-isopropylideneadenosine

Abstract

Bromination of the title compound 1 with bromine in phosphate buffer has led to 8-bromo-N⁶, N⁶-dimethyl-2′,3′-0-isopropylidene-adenosine (2) and 2′,3′-0-isopropylidene-N⁶-methyladenosine (3). Under similar conditions, compound 2 gave 8-bromo-2′,3′-0-isopropylidene-N⁶-methyladenosine (4). The transformations 1 → 3 and 2 → 4 represent biomimetic models of in vivo N⁶-demethylation of antibiotic puromycin.     

1H NMR Assignments and Conformational Analysis of the Oligoribonucleotides CA,CAU, CAUG,ACAUG, and UCAUG: Observation of Pyrimidine H5-H1′ Long-Range Scalar Couplings

Abstract

Conformational analysis and ¹H NMR spectral assignments have been carried out using COSY and RELAY methods for a series of related oligoribonucleotides including two pen- tamers with 5′-dangling bases. Intraresidue long-range five bond scalar coupling was observed between pyrimidine H5 and H1′ protons in the COSY-45 spectra and this feature was useful for both assignment purposes and conformational analysis. The ribose ring conformations were predominantly C3′-endo with the C2′-endo population increasing at the 3′-terminus. The 5′-dangling bases were not stacked efficiently, exhibiting lower % C3′-endo values than their 3′-nearest neighbors. Backbone torsion angle populations, β′, γ⁺, ε′, were determined using ′H-′H, ′H-³¹P, and ¹³C-³¹P coupling constants. From β′ and γ⁺ populations the U3-G4 step in CAUG was found to be less efficiently stacked than the C1-A2 and A2-U3 steps. This observation in solution is consistent with the fiber diffraction A-RNA model (S. Arnott, D.W.L. Hukins, S.D. Dover, W. Fuller and A.R. Hodgson, J. Mol. Biol. 81, 107-122, 1973) which also predicts poor stacking in a U-G dinucleotide. The ε′ populations were >65% for all C3′- O3′ bonds and consistent with a right-handed A-RNA helix.     

Germacranolides from Viguiera microphylla

Three new germacranolides, including two heliangolides (niveusin C-2′,3′-epoxide and 1,2-dehydroniveusin C-2′,3′-epoxide) and a germacrolide (3β-hydroxy-8β-epoxyangeloyloxycostunolide-1β,10α-epoxide) were isolated from Viguiera microphylla. Their structures were established by spectroscopic analyses, including extensive ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations. X-ray diffraction analysis confirmed the structure of niveusin C-2′,3′-epoxide.     

Neolignans from mezilaurus itauba

The wood bark of Mezilaurus itauba afforded in addition to seven known neolignans, three new compounds rel-(7R,8R,1′S,3′S)-Δ^5′,8′-5′-methoxy-3,4-methylenedioxy-1′,2′,3′,4′-tetrahydro-2′,4′-dioxo-7.3′,8.1′-neolignan, rel-(7S,8S,1′S, 2′S, 3′R, 4′S)-Δ^8′-2′,4′-dihydroxy-3,4-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan and rel-(7S,8S)-Δ^8′-6′-hydroxy 5′-methoxy-3,4-methylenedioxy-7·O·2′,8.3′-neolignan. The latter compound has been detected previously in Aniba terminalis. The structures were elucidated by spectroscopic methods and comparison with related compounds.     

Caldensinic acid,a prenylated benzoic acid from Piper caldense

The CH₂Cl₂ and MeOH extracts from leaves of Piper caldense were subjected to chromatographic separation procedures to afford the new prenylated benzoic acid, caldensinic acid (3-[(2′E,6′E,10′E)-11′-carboxy-3′,7′,15′-trimethylhexadeca-2′,6′,10′,14′-tetraenyl]-4,5-dihydroxybenzoic acid) whose structure was determined by spectral analysis, mainly NMR (¹H, ¹³C, HSQC, HMBC) and ESI-MS. The natural compound and derivatives displayed antifungal activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum by direct bioautography.     

Synthesis,Protonation Behavior,Conformational Analysis,and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

Abstract

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through ¹H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.     

Synthesis and Reactions of Some 3′,4′-Unsaturated 2′,3′-Secouridine Analogues

Abstract

2′,3′-Dibromo-2′,3′-dideoxy-5′-O-trityl-2′,3′-secouridine (8) with sdKF gave the 3′,4′-didehydro-2,2′-anhydro nucleoside 9, which was deprotected to 10. Hydrolysis of 9 gave 3′,4′-didehydro-3′-deoxy-5′-O-trityl-2′,3′-secouridine (11a). Similarly, compound 9 with pyridinium halides gave the corresponding 2′-deoxy-2′-halo nucleosides (11b-d). Compound 11d with azide ion gave 2′-azido analogue 11e. Compound 9 with an excess amount of azide ion gave the 2′-azido triazole (13).     

Crystal Structure of 1-(2′,3′,5′-Tri -O-Acetyl-β-D-ribofuranosyl)-3-acetoxy-2-pyridone: An Antitumour Agent

Abstract

1–C3′, 3′, 5′–Tri—O—acetyl— β—D—ribof uranosyll)—3—acetoxy —2—pyridone,crystallised in space group P2 with z=2 and cell parameters a=12. 446(2), b=10. 415(2), c=7. 600(2) A, β=03. 3O(4). The structure was solved by direct methods and refined by full—matrix least—squares to a final R value of 0·251 for 1847 observed reflections. The sugar—pucker is found to be ³ECC3′ endo) with P = 17.7° and x_CN=170. 2(2)° in the range. The C4′-C5′ conformation is gauche minus. Because of the absence of H—bond donor atoms. the crystal structure is stabilised by a network of C-H—-O close contacts. No base stacking is observed.     

Stereoselective 5′-Monodeuterated Ribonucleoside Phosphoramidites as Tools for Conformational Studies of RNA by the NMR Approach

Abstract

The analysis of NMR spectra of DNA and RNA, in particular, homo- and heteronuclear vicinal coupling constants of the nuclei of the sugar-phosphate backbone, can provide important information about the conformation of macromolecules¹. For example, 5′H - P coupling constants allows us to obtain a value of β torsional angle, 5′H-4′H - γ, whereas 3′H-P constant gives the angle ?. Unfortunately, due to the complex structure of H5′, H4′ and H3′ multiples in moderate and large RNA fragments (>15 nucleotides), it is very difficult to assign signals and extract accurate structural data.     

Detection of a 3′, 5′-cyclic-AMP phosphodiesterase activity in the lichenized fungus Evernia prunastri

Abstract

A 3′, 5′-cyclic-AMP phosphodiesterase (PDE) was detected and measured in the lichen Evernia prunastri. The percentage of hydrolysis of tritiated 3′, 5′-cyclic-adenosine monophosphate ([³H]-cAMP) and 3′, 5′-cyclic-guanosine monophosphate ([³H]-cGMP) by the PDE enzyme into tritiated 5′-adenosine-monophospahte ([³H]-AMP) and tritiated 5′-guanosine-monophospahte ([³H]-GMP) was measured by treating the PDE products with a 5′-nucleotidase enzyme present in snake venom. The lysate fraction (L) (plasma membranes and cell walls) and the supernatant (S) (soluble fraction of the cells) were tested. In both fractions, competition of unlabelled cAMP, but not unlabelled cGMP, was revealed. Specific competitive PDE inhibitors such as IBMX inhibited enzymatic activity. Although it is thought that in this species cAMP is regulated by red/far red light through PDE activity, this is the first report that seems to suggest the presence of a PDE activity specific for cAMP in lichenized fungi. However, this work is at a preliminary stage and despite the high levels of enzymatic activity with cAMP found in both fractions, data are still insufficient to state the absolute specificity for this nucleotide.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Synthesis and Evaluation of Antimicrobial Activity of Some Pyrimidine Glycosides

Reaction of ethyl 4-thioxo-3,4-dihydropyrimidine-5-carboxylate derivatives 1a,b and ethyl 4-oxo-3,4-dihydropyrimidine-5-carboxylate 1c with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in KOH or TEA afforded ethyl 2-aryl-4-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosylthio or/ oxy)-6-methylpyrimidine-5-carboxylate 6a-c. The glucosides 6a and 6b were obtained by the reaction of 1a and 1b with peracetylated glucose3 under MW irradiation. Mercuration of 1a followed by reaction with acetobromoglucose gave the same product 6a. The reaction of 1a-c with peracetylated ribose 4 under MW irradiation gave ethyl 2-aryl-4-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosylthio)-6-methylpyrimidine-5-carboxylate 8a–c. The deprotection of 6a–c and 8a–c in the presence of methanol and TEA/H₂O afforded the deprotected products 7a–c and 9a–c. The structure were confirmed by using ¹H and ¹³CNMR spectra. Selected members of these compounds were screened for antimicrobial activity.     

Mutational tests of the NMR-docked structure of the staphylococcal nuclease–metal–3′,5′-pdTp complex

In the X-ray structure of the staphylococcal nuclease–Ca²⁺ ?3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca²⁺, and Mn²⁺ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca²⁺ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca²⁺-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.     

Conformational Analysis of the Deoxyribofuranose Ring in DNA by means of Sums of Proton-proton Coupling Constants: A Graphical Method

Abstract

A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-pro ton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as Σ1′ (= J1′2′ + J1′2″), Σ2′ (= J1′2′ + J2′3′+J2′2″), Σ2″ (= J1′2″ + J 2″3′ + J2′2″) and Σ3′ (= J2′3′ + J2″3′ + J3′4′). These sums of couplings correspond to the distance between the outer peaks of the H1′, H2′, H2″ and H3′ {³¹P} resonances, respectively, (except for Σ2′ and Σ2″ in the case of a small chemical shift difference between the H2′ and H2″ resonances) and can often be obtained from ¹H-NMR spectra via first-order measurement, obviating the necessity of a computer-assisted simulation of the fine structure of these resonances. Two different types of graphs for the interpretation of the coupling data are discussed: the first type of graph serves to probe as to whether or not the sugar ring occurs as a single conformer, and if so to analyze the coupling data in terms of the geometry of this sugar ring. In cases where the sugar ring does not occur as a single conformer, but as a blend of N-and S-type sugar puckers, the second type of graph is used to analyze the coupling data in terms of the geometry and population of the most abundant form.

It is shown that the latter type of analysis can be carried out on the basis of experimental values for merely Σ1′, Σ2′ and Σ2″, without any assumptions or restrictions concerning a relation between the geometry of the N- and S-type conformer. In addition, the question is discussed as to how insight can be gained into the conformational purity of the sugar ring from the observed fine structure of the H1′ resonance. Finally, a comparison is made between experimental coupling data reported for single-stranded and duplex DNA fragments and covalent RNA-DNA hybrids on the one hand and the predicted couplings and sums of couplings presented in this paper on the other hand.     

Synthesis,Solution Conformation and Biological properties of 2′,3′-Dideoxy-3′-fluoro-D-erythro-pentofuranosides of 2-Thiouracil and 2-Thiothymine

Abstract

The synthesis of the α- and β-anomers of 2′,3′-dideoxy-3′-fluoro-2-thiouridine and 2′,3′-dideoxy-3′-fluoro-2-thiothymidine via Lewis acid catalysed nucleoside condensation is described. High resolution ¹H NMR data, solution conformations and biological properties are also presented.     

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.