C. Glemarec

Structure of a hepatotoxic pentapeptide from the cyanobacterium Nodularia spumigena

The structure of a hepatotoxic peptide from the cyanobacterium Nodularia spumigena was determined using 1D and 2D proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The toxin was a cyclic pentapeptide (mol. wt 824.5) with the structure cyclo-(beta-methylisoAsp-Arg-Adda-isoGlu-N-methylde hydrobutyric acid) (Adda: 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid).

An efficient synthesis of y-nucleoside (wyosine) by regiospecific methylation of n4-desmethylwyosine using organozinc reagent

The reaction of the organozinc reagent, produced by the reaction of CH2I2 + Zn(C2H5)2 + glyme in diethylether at 20 °C, regioselectively methylates the N4-nitrogen of N4-desmethylwyosine-triacetate 3 to give pure wyosine-triacetate 1e in 76% isolated yield; no trace of the isomeric N5 methylated product is found in the latter reaction. Synthesis of a new congener of Y-nucleoside 1f is also reported for the first time from compound 5 in a high overall yield using a similar procedure.

Spectroscopic, kinetic and semiempirical molecular orbital studies on 8-amino-, 8-methylamino- & 8-dimethylamino-adenosines

Summary: Kinetic studies on acidic depurination of 8-aminoadenosine, 8-methylaminoadenosine, and 8dimethylaminoadenosine show their relative rates are respectively 2.7,2 and 429,fold with respect ta adenosine. Structural consequence of 8-amino, 8-methylamino or 8-dimethylamino group in the g-substituted purine nucleosides have been therefore investigated in order to delineate the injluence of these d-amino substituents on the relative rate of cleavage of glycosyl bond under acidic condition by both IsN- and IH-NMR spectroscopy in neutral and acidic solutions. ISN-NiUR studies showed that the relative amount of protonation at Nl in 8-amine adenosine and B-methylaminoadenosine are 66% (4470 N’H+) and 8.5% (15% N’H+), respectively, while it is 96% (4% N7H+) in case of 8-dimethylaminoadenosine. IH-NMR studies also showed some differences in conformation of sugar moiety of nucleosides in acidic solution in comparison with their con

The self-cleavage of lariat-RNA

ormations in neutral solution. Semiempirical Molecular Orbital calculations have been used to throw light on steric and electronic factors dictated by a-amino, 8-methylamino or 8-dimethylamino group across CB-N8 bond that control the stabilization of N7H+ versus NlH+ species. Result from these Semiempirical Molecular Orbital calculations have been subsequently assessed with those obtained by IsN-NMR studies. Successful rational design of new analogues of nucleosides which should interfere specifically with the biosynthesis of DNA or RNA in cancer cells or virus- or parasite-infected cells requires that the effect of specific structural modification in these analogues on the chemical reactivities, hydrolytic stabilitities, and conformational properties in solution are clearly defined. In this respect, we considered 8-aminoadenosine, 8methylaminoadenosine, and 8-dimethylaminoadenosine as useful target nucleosides because of the following reasons. Lipophilic derivatives of cytosine and guanosine have been shown to form Watson-Crick type base pairs in nonaqueous solvents over a wide temperature range suggesting that the monomers can be used to understand and possibly to predict structures of polymeric nucleic acids’, since low dielectric solvents mimic the dielectric environment of the interior of a nucleic acid double helix. Lipophilic derivatives of 8-amino-2’deoxyadenosine and 8-methylamino-2’-deoxyadenosine are expected to form both Watson-Crick and Hcogsteen type base pairs with lipophilic thymidine derivative in non-polar solution while 8-dimethylamino-2’deoxyadenosine should not. Our initial studies on the base-pairing abilities of lipophilic 8-amino-2’deoxyadenosine derivative with lipophilic thymidine derivative in CDC13 have shown that their hydrogenbonded complexation in 1: 1 and 1: 2 molar stoicheiometries at -20 “C are differentx. These studies have indicated that oligonucleotide based on 8-amino-2’-deoxyadenosine or 8-methylamino-2’-deoxyadenosine should form intermolecular DNA triplexes which have been shown to be important because of their potential role in chromosome mapping3 and in antisense gene therapfl. Our unpublished work has shown that the glycosidic bond of 8-amino-2’-deoxyadenosine [8-A-2,&I] and 8-methylamino-2’-deoxyadenosine [8-mA-2’-dA] are -10 times more acid-labile than that of the parent 2’-deoxyadenosine, which suggests that no acid-labile protecting group can be used in the synthesis of [8-A-2’-dA 18 & [S-mA-2’-dA]g. These observations prompted us to study the structural properties of 8-amino- & 8-alkylamino substituted adenosine and guanosine derivatives in

Solution structure of branched U3′p5′A2′p5′G3′p5′C and its comparison with A2′p5′G3′p5′U by 500 MHz NMR spectroscopy

Abstract Lariat-RNAs 3 and 4 undergo site-specific self-cleavage reaction at the G 3 → C 6 /U 7 phosphodiester bond by the nucleophilic attack of 2′-OH of G 3 sugar moiety to its 3′-phosphate to give 5′-hydroxyl terminal at C 6 or U 7 and 2′,3′-cyclic phosphodiester of G 3 whereas lariat-tetramer 1 , pentamer 2 , the cyclic-A(2′→5′)G-tetramer 5 and the cyclic-A(3′→5′)G-tetramer 6 are completely stable. The lariat-RNAs 3 and 4 are the smallest RNA known to undergo self-cleavage which is reminiscent of the RNA-hammerhead (Ribozyme) activity. The geometry of the cleavage-site in 3 and 4 has been defined by full conformational analysis by NMR and molecular dynamics calculation in water.

Synthesis of 2′,3′-cis-fused pyrrolidino-β-D-nucleosides and their conformational analysis by 500 MHz 1H-NMR

Abstract In this study, the 1H-1H, 1H-13P and 13C-31P coupling constants of the branched RNA tetramer 2 have been measured at two temperatures to obtain detailed information about its backbone conformation. Evaluation of these coupling constants by Karplus-Altona algorithm shows the decrease of populations of γ+ and βt upon temperature-increase for the branch-point A and 3′-terminal C residues, which have been attributed to a destacking along the U3′→5′A3′→5′C stacked axis in the tetramer 2. In accordance with this observation, it has been clearly established that γ+ and β+ populations of constituent 2′→5′-linked guanosine nucleotide is rather insensitive to temperature-change. The NOEs seen at 270 MHz between AH8 with UH6, and AH2 with CH6 also support that the tetramer 2 stacks along the U3′→5′A3′→5′C axis. The NOEs observed at 270 MHz between CH6 with GH8, and UH6 with GH8 suggest also a spatial proximity between 5′-terminal U and 2′-terminal G, and 3′-terminal C and 2′-terminal G residues. These observations have led us to propose a two-state model for the tetramer 2. On the other hand, detailed temperature-dependent measurements of 1H-1H, 1H-31P and 13C-31P coupling constants and chemical shifts of analogues of the branched trimer 1 in this laboratory and elsewhere have shown that the molecular conformation of the branched trimer 1 is governed by A2′→5′G stack. The introduction of a 5′-terminal uridine residue in trimer 1 to tetramer 2 shifts the molecular conformation from an A2′→5′G stack in the trimer 1 to a A3′→5′C stack in the tetramer 2. This is a new example of 5′-terminal residue promoted conformational transmission.

Conformational studies of thymidine dimers containing sulfonate and sulfonamide linkages by NMR spectroscopy

Abstract The unique “off-template” stereoselectivity in the intramolecular free radical cyclization of 3-aza-5-hexenyl endocyclic radical has been demonstrated for the first time through the synthesis of 2′,3′- cis-fused pyrrolidino-β-D-nucleosides, 19a, 20a, 21a, 22a, 23a, 35a, 36a, 37a and 38a, which are not hithertofore available by any known procedures. The 2′-linked chiral carbon (Cc) in 19a, 20a, 21a and 22a has shown 25:1 to 10:1 stereoselectivity depending upon the bulk of its substituent. The 3′-linked chiral carbon (Cc) in 35a, 36a and 37a, on the other hand, has shown only 4:1 to 2:1 stereoselectivity. Finally, a full conformational analysis of 2′,3′-cis-fused pyrrolidino-β-D-nucleosides 19a, 20a, 21a, 22a, 23a, 35a, 36a, 37a and 38a is reported using 1H-NMR at 500 MHz. The solution geometry of the furanose and the pyrrolidine rings were studied on the basis of vicinal proton-proton coupling constants using the concept of pseudorotation. The furanose rings in 19a – 23a have a geometry biased toward a South-type conformation [70 – 81 % S, 126°

N23-ethenoguanosine and IA′-metamorphosine: 5N NMR Spectroscopy and elucidation of physico-chemical properties by kinetic and equilibrium measurements

Abstract The conformations of 3′-azido-terminated-sulfonate-dimer 1 and 3′-amino-terminated-sulfonamide-dimer 2 are characterized by the following features: (1) The 5′-terminal nucleoside moiety of 1 has a S-type sugar (87% S), a staggered γ+ (rotamer across the C4′C5′ bond (65%) and an anti orientation of the base about the glycosidic bond. The 5′-terminal nucleoside moiety of 2 has an almost equal population of S and N conformations, a staggered γ+ rotamer (69 %) and an anti orientation of the base. (2) The 3′-terminal nucleoside moieties of 1 and 2 are in ∼50% N ⇄ S equilibrium and the γt conformer is the most populated. A comparison of the conformational properties of 1 and 2 with the natural thymidyl(3′ → 5′)thymidine [d(TpT)] 3, thymidylyl-(3′ → 5′)-5′-thio-5′-deoxythymide d(TpST)5 4 and thymidinylacetamido-[3′(O)→5′(C)]-5′-deoxythymidine NH2d(TcmT)5 5, show the following characteristics: (i) The conformational preference of the sugar ring is partially determined by the gauche effect. This means that the more polar the C3′X bond due to the electronegative character of the 3′-α-X substituent, the more than N ⇌ S equilibrium is biased toward the S-type conformation: 3′-O-S > 3′-O-H > 3′-N3 > 3′-NH2. (ii) The conformation about the C4′C5′ bond (γ) is also influenced by the gauche effect based on the nature of the 5′-substituent and by the ability of the 5′-substituent to form hydrogen bonding with the H6 of thymine. Thus, the population of the γ+ conformer follows the order: 5′-O > 5′-N > 5′-S > 5′-C. (iii) The C5′C6′ bond has a slight preference for the βt conformation (56 % βt in 1 and 58 % βt in 2), while in natural d(TpT) 3, the C5′O5′ bond accounts for 83% of βt conformation. Upon substitution of the 5′-oxygen by 5′-sulfur, as in d(TpST) 4, the population of βt conformer was found to decrease to 57 %. This decrease in the βt population in 1, 2 and 4 is a result of the reduced polarity of the 5′CX [X = CH2 in 1 and 2 and X  S in 4] in comparaison to the 5′CO bond in 3, which weaken the gauche effect.

Fluorescence Properties of Y-Nucleoside Derivatives

Abstract Tautomerism, protonation and electronic properties of the base moieties of IA′-metamorphosine (1a), N2,3-ethenoguanosine (2a) and O6-benzyl-N2,3-ethenoguanosine (3a were investigated by 15N NMR spectroscopy. pKa values of the same compounds were determined spectrophotometrically, and hydrolytic stability of the N-glycosidic bond was studied at various hydronium ion concentrations. The base stacking ability and metal ion complexation of N2,3-ethenoguanosine and its 9-ethyl counterpart (7) were elucidated by phase distribution and potentiometric measurements.

The application of Uppsala ''NMR-window'' concept for conformational analysis of biologically functional DNAs and RNAs by high-field NMR

Abstract Modified-base and modified N3-(β-D_-ribofuranoside) derivatives of the Y base exhibit subnanosecond fluorescence decays. Attachment of groups to the “unnatural” N1 base position produces compounds showing dominant 7 - 10 ns decays. These spectral properties have been compared with those of the free Y base and suggest that the free Y base exists mainly as the N1-H tautomer.