Solid-supported synthesis of 5'-mRNA CAP-4 from Trypanosomatids

The unique structure of 5' mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5'-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn(2+) as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl(2).     

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.     

Conformational analysis of the deoxyribofuranose ring in DNA by means of sums of proton-proton coupling constants: a graphical method

A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-proton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as sigma 1' (= J1'2' + J1'2'), sigma 2' (= J1'2' + J2'3' + J2'2'), sigma 2' (= J1'2' + J2'3' + J2'2') and sigma 3' (= 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 sigma 2' and sigma 2' 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 sigma 1',sigma 2' and sigma 2', 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.     

Conformation of mononucleotides and dinucleoside monophosphates. P[H] and H[H] nuclear Overhauser effects.

The phosphorus-proton nuclear Overhauser effect (NOE) was used to investigate the quantitative distribution of rotamers about the C3'--O3' bond (phi') of 3'-AMP and 2',3'-cyclic-CMP and the C4'--C5', C5'--O5' bonds (psi, phi) of 5'-AMP. Phosphorus-proton and proton-proton NOE's were used to provide a qualitative insight into the backbone conformation and the glycosyl angle torsions of adenosylyl-(3' leads to 5')-adenosine (ApA). The major psi rotamer in 5'-AMP is the 60 degree (gg) form, while the major phi rotamer is the 180 degrees (g'g') form. The constrained model, 2',3'-cyclic-CMP, manifests the C3'endo furanose pucker predominantly. The results from these two models are consistent with nuclear magnetic resonance (NMR) J coupling analyses. The phi; distribution of 3'-AMP is dominated (77%) by the 180 degrees g- rotamer. The 3'-AMP results are consistent with phosphorus-hydrogen coupling constant analyses, but do not accord with phosphorus-carbon coupling constant results. The phosphorus-proton NOE reveals that the phosphorus of ApA occupies a region of conformation space not seen in 5'-AMP. The proton-proton NOE on APA shows a significant portion of syn rotamer in both X distributions and detects a cross-purine ring interaction consistent with base stacking known to exist in this system.     

Design and synthesis of a vialinin A analog with a potent inhibitory activity of TNF-α production and its transformation into a couple of bioprobes

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.     

A proton magnetic resonance investigation of the glycosyl torsion angle of uracil nucleosides and nucleotides.

The use of line-shape decomposition techniques permitted the small 5-bond (5-J51') and 4-bond (4-J61') proton-proton coupling constants of a series of uracil nucleosides and nucleotides to be determined accurately. From an analysis of these coupling constants we have determined that the uracil base is in a predominantly anti conformation in aqueous solution and the mean position is not substantially altered by phosphate substitution at the 2', 3', or 5' positions, by changing the furanose stereochemistry from a ribose to a deoxyribose or an arabinose, or by an increase in temperature of 43 degree C.     

Synthesis of 5'(3')-O-amino nucleosides

Synthetic methods leading to 5'(3')-O-amino nucleosides have been developed in an effort to prepare derivatives that may have antitumor or antiviral activities. They are based on ring opening of O2,5'-cyclonucleosides with the N-protected hydroxylamines and dehydrative coupling of 5'(3')-O-unprotected nucleosides with N-hydroxyphthalimide.     

Utilization of Splicing Elements and Polyadenylation Signal Elements in the Coupling of Polyadenylation and Last-Intron Removal

Polyadenylation (PA) is the process by which the 3' ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3' splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5' splice site in the presence of a viable polypyrimidine tract and the 3' splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5' splice site) within the last exon can negatively effect both PA and splicing; hence, a 5' splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.     

Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A(C)', A(C), and B(C)) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A(C)' complex is formed by 30 s after mixing, the A(C) complex is formed between 1 and 2 min after mixing, and the B(C) complex is formed by 2 to 3 min after mixing. A(C)' is a precursor of A(C), and the A(C)' and/or A(C) complex is a precursor of B(C). Of the three complexes, B(C) appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A(C)' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A(C)' complex is dependent on the polypyrimidine tract. The transition from A(C)' to A(C) appears to require an intact 3'-splice site. Formation of the B(C) complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A(C)' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B(C) complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.     

Efficient synthesis of gamma-DDB

Synthesis of gamma-DDB, which is another family member of gamma-DDB (dimethyl 4,4(')-dimethoxy-5,6,5('),6(')- dimethylenedioxybiphenyl-2,2(')-dicarboxylate), is described. The unsymmetric isomer (gamma-DDB) was constructed by a linker-directed intramolecular Ullmann coupling reaction, followed by the cleavage of the linker and re-esterification.     

13C-NMR of ribosyl ApApA, ApApG and ApUpG

The chemical shifts as well as the 13C-31P coupling constants of the carbon-13 nuclei in single-stranded ApApA, ApApG, and ApUpG are sensitive to sequence and temperature. ApApA and ApApG have similar properties with large shielding (up to 1.7 ppm) of many of the base carbons upon decreasing the temperature from 70 degrees C to 11 degrees C; the base carbons have smaller shielding changes in ApUpG. Large shielding and deshielding effects are observed for the 1', 3', 4' and 5'-carbons over this temperature range. Analysis of the 13C-31P couplings measured at the 4' ribose carbons show that the population of the anti rotamer about O5'-C5' varies from 98 to 75%, and is higher in ApApA and ApApG than in ApUpG. The CCOP coupling data at 2' and 4' is consistent with a blend of the -antiperiplanar/-synclinal nonclassical rotamers about the C3'-O3' bond, varying from 89/11% in ApApG to 55/45% in ApUpG. The coupling and chemical shift data support the thesis that ApUpG is stacked much less than the other two molecules. The stacked forms of all three trinucleotides is most easily interpreted by a standard A-RNA model. It is not necessary to invoke the "bulged base" hypothesis [Lee, C.-H. and Tinoco, Jr., I. (1981) Biophysical Chemistry 1, 283-294; Lankhorst, P.P., Wille, G., van Boom., J.H., Altona, C., and Haasnoot, C.A.G. (1983) Nucleic Acids Research 11, 2839-2856] to explain the contrast in 13C spectroscopic properties of ApUpG in comparison to ApApG and ApApA.     

Stereospecific assignment of H5′ and H5″ in a (5′R)-/(5′S)-deuterium- labeled DNA decamer for3 JHH determination and unambiguous NOE assignments

The stereoselective deuterium labeling at the 5' methylene protons of the ribose ring recently developed by Kawashima et al. [1995, Tetrahedron Lett., 36, 6699–6700] enabled the assignment of pro-R and pro-S protons at the 5' position. The deuterium-labeled nucleotides, [(5'S)-²H]- and [(5'R)-²H]-diastereomers, in an approximate ratio of 2:1, were incorporated in the decamer 5'-d(GCATTAATGC)-3'. Thus, both pro-R and pro-S methylene proton signals without geminal coupling appeared in the NOESY and DQF-COSY spectra. Complete stereospecific assignments and simplified spin systems enabled the determination of 15 ³J coupling constants between H4' and H5'/H5", and the unambiguous assignment of 135 NOESY cross peaks originating from H4'/H5'/H5" resonances.     

New analogs of acyclovir substituted at the side chain

A series of novel analogs of acyclovir, substituted with an alkyl (methyl, ethyl, n-butyl) or phenyl group at the positions 1', 4', and/or 5', has been obtained in a direct one-pot coupling reaction of guanosine and the respective 1,3-dioxolanes. The new acyclonucleosides were essentially inactive in antiviral (HSV, VV, VSV, HBV) evaluation in vitro.     

Synthesis of (3'-5'),(2'-5')-linked di- and tri-adenylyl methylphosphonate analogs.

Stereoselectivity was found during the coupling reaction, to form 2',5'- and 3',5'-linked di- and triadenylyl methylphosphonate. The configuration of phosphorus was determined by 1HNMR NOE.     

Investigation of the mechanism of the synthesis of oligonucleotides. IX. 31P NMR spectra of the active dinucleotide derivatives and their analogs.

The interaction of 3'-O-acetyldithymidilate (pdTpdT(Ac)), thymidine-3',5'-diphosphate (pdTp) and thymidine-3'-phenyl-phosphate-5'-phosphate (pdTpPh) with 2,4,6-triisopropylbenzene sulphonyl chloride (TPS) and N,N'-dicyclohexylcarbodiimide (DCC) in pyridine and dimethylformamide (DMF) was studied by pulsed NMR spectroscopy on phosphorus nuclei. Thymidine cyclic 3',5'-pyrophosphate and dimeric pyrophosphate derivatives were shown to be the main products of the reaction of pdTp with TPS and DCC. The former shows spin AB-system with the unusually large spin-spin coupling constant about 28Hz upfield to the signals of the dimeric pyrophosphates in NMR spectrum. Analogous spin AB-systems with large spin-spin coupling constants (up to 32 Hz) were observed in the spectra of the reaction mixtures of pdTpdT(Ac) with TPS or DCC and of pdTpPh with TPS. These spin AB-systems were ascribed to 3',5'-cyclic pyrophosphate derivatives of pdTpdT(Ac) and pdTpPh.     

Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.     

Conformational studies of some 2':3'-cyclic mononucleotides in solution by different nuclear-magnetic-resonance methods

The conformations of the 2':3'-cyclic mononucleotides of adenosine and cytidine in deuterium oxide has been studied at pH 2.3, using lanthanide ions as paramagnetic nuclear magnetic resonance (NMR) probes. It was not possible to find any single conformation for these molecules which accounts for the observed shift and relaxation data. This situation is in agreement with the interpretation of vicinal 1H-1H and 1H-31P coupling constants, which indicate that the ribofuranose and cyclic phosphate rings are in rapid equilibrium between different puckered forms. The interpretation of the lanthanide data in terms of an equilibrium between different conformations give average rotamer populations in good agreement with the coupling constant analysis. The conformations of these systems in aqueous solutions were found to be more flexible than in the solid state, where rigid planar ribofuranose rings have been observed. Adenosine 2':3'-monophosphate differs from cytidine 2':3'-monophosphate at the glycosidic link.     

The conformation of the d(ACCCGGGT) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)2 have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D2O at 12 degrees C. The observed NOE's between the base protons and their own H2' protons and between the base protons and the H2' protons of the 5' adjacent nucleotide and the observed coupling constants between the deoxyribose 1' and 2',2' protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

Electrotonic Transmission of an Action Potential between Two Separated Internodal Cells of Chara through a Bridge

Two separated internodal cells of Chara braunii were broughtinto contact with each other longitudinally at their ends andconnected by another pathway composed of a metal bridge beyondthe region of intercellular contact. A conducted action potentialthat arrived at one foot of the bridge electrotonically depolarizedthe other foot of the bridge in the connected cell. The electriccoupling ratio (0.07?0.03), the ratio of the change in the membranepotential of one cell to that of the other cell, was too smallto allow transmission of an action potential. Two cells wereplaced in parallel and connected with two liquid bridges orpools, a' and b'. When the action potential of one cell wasconducted through one connecting pool (pool a)', the other cellwas depolarized electrotonically by the action current via theother connecting pool (pool b'). The coupling ratio was increasedto 0.26?0.07 by the solution bridge, but transmission of theaction potential was rarely observed. Application of 1 mM KC1to pools a' and/or b' slightly improved the frequency of transmissionof the action potential. When pool b' contained 5% urethane,the coupling ratio increased to 0.31?0.08 and transmission ofthe action potential was frequent. (Received August 24, 1989; Accepted March 14, 1990)     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.