2'-modified-2-thiothymidine oligonucleotides

[reaction: see text] Oligonucleotides with novel modifications, 2'-O-[2-(methoxy)ethyl]-2-thiothymidine and 2'-deoxy-2'-fluoro-2-thiothymidine, have been synthesized. These modified oligonucleotides exhibited very high thermal stability when hybridized with complementary RNA. 2-O-(2-Methoxy)ethyl-2-thiothymidine modified oligonucleotide phosphodiesters showed enhanced resistance toward nucleases (t(1/2) > 24 h), but 2'-deoxy-2'-fluoro-2-thiothymidine modified oligonucleotide phosphodiesters showed limited stability to nucleotytic degradation.     

Conformational analysis of furanose rings with PSEUROT: parametrization for rings possessing the arabino,lyxo, ribo,and xylo stereochemistry and application to arabinofuranosides

The solution conformation of a furanose ring can be assessed through PSEUROT analysis of three-bond (1)H-(1)H coupling constants ((3)J(HH)) of the ring hydrogens. For each coupling constant, PSEUROT requires two parameters, A and B, which are used to translate the H[bond]C[bond]C[bond]H dihedral angle predicted from the (3)J(HH) into an endocyclic torsion angle from which the identity of the conformers can be determined. In this paper, we have used density functional theory methods to generate a family of envelope conformers for methyl furanosides 1-8. From these structures, A and B were calculated for each H[bond]C[bond]C[bond]H fragment. In turn, the values of these parameters for the arabinofuranose ring were used in PSEUROT calculations to determine the conformers populated by monosaccharides 1 and 2 as well as the furanose rings in oligosaccharides 9-15. The results of these analyses are consistent with the low-energy conformers identified from previous computational and X-ray crystallographic studies of 1 and 2.     

2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides: synthesis,stereospecificity, some chemical transformations,and conformational analysis

The synthesis of methyl 5-O-benzoyl-2-chloro-2,3-dideoxy-3-fluoro-beta-d-ribofuranoside (5) and its use as a glycosylating agent for persilylated thymine, N(6)-benzoyladenine, and N(4)-benzoylcytosine are described (Scheme 1). The 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides 10-12 synthesized were transformed to 2',3'-dideoxy-3'-fluoro-alpha- and -beta-d-erythro-pentofuranoside nucleosides of thymine (13a,b), adenine (14a,b), and cytidine (15a,b) by treatment with tributyltin hydride in the presence of alpha,alpha'-azobisisobutyronitrile (Scheme 2). Treatment of 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides with 1 M MeONa/MeOH under reflux for 1-5 h afforded 2',3'-didehydro-2',3'-dideoxy-2'-chloro-d-pentofuranosyl nucleosides as the principal products (47-81%) of the reaction, along with recovered starting nucleoside (11-33%) (Scheme 3). Easy HF elimination was also observed in the case of the 2'-azido-2',3'-dideoxy-3'-fluoro-beta-d-ribofuranosides of thymine (17) and adenine (20) (Scheme 3). The role of conformational peculiarities of 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides as well as of 17 and 20 in the observed exclusive elimination of HF is discussed. The conformational analysis of a rather broad palette of 2,3-dideoxy-3-fluoro-2-(X-substituted)-d-ribofuranosides was performed with the aid of the PSEUROT (version 6.3) program, using (i) the recently reparametrized Karplus-type relation (Chattopadhyaya and co-workers. J. Org. Chem. 1998, 63, 4967) and (ii) empirical bond angle correction terms suggested by us. The predictive power of the Brunck and Weinhold model (J. Am. Chem. Soc. 1979, 101, 1700) of the gauche effect between atoms and groups as a conformational driving force acting upon the pentofuranose ring is explored. Their model invokes maximum antiperiplanar sigma <--> sigma stabilization when the donating bond is the least polar one and the acceptor orbital is at the most polarized bond and is found at least as satisfactory, and in various specific cases more so than, as rationalizations on the basis of the preference of the gauche vs the trans conformation of two vicinal electronegative substituents (Wolfe. Acc. Chem. Res. 1972, 5, 102).     

Conformation, Inversion Barrier, and Solvent-Induced Conformational Shift in Exo- and Endo/Exo-Calix[4]arenes

Calixarenes 4a and 4b having hydroxyl groups in endo and exo positions and the ethanediyl-bridged exo-calixarene 5a were synthesized by a stepwise strategy. Single-crystal X-ray structures were obtained for 4a and for the exo-calixarene 3d, showing the molecules to exist in the 1,2-alternate conformation which is also found for 4a,b in solution. The inversion barriers of 4a and 4b (10.3 and 10.8 kcal mol(-1)) are similar to that determined for the endo-dihydroxycalixarene 12, indicating that the additional intramolecular hydrogen bond between the exo OH groups does not decrease the flexibility of the molecule. In CDCl(3) solution exo-calixarene 5a adopts a 1,2-alternate conformation with the methyl group at the bridge located in an axial position, while in DMSO-d(6) the conformation adopted is the partial cone. Similar solvent-induced conformational shifts were found for the exo-calixarenes 3b and 3d. MM3 calculations predict that the cone form is the lowest energy conformation of 4 and the exo-calixarenes 3 and 5. The calculations suggest that the conformational preferences of the methyl group at the bridge for either the axial or equatorial positions are in large part determined by the repulsive steric interactions with the hydroxyl groups. The inversion barrier of 4b is satisfactorily reproduced by calculations, which indicate that the rotation of the exo rings is less energetically demanding than the rotation of the endo rings.     

Partially O-alkylated thiacalix[4]arenes: synthesis, molecular and crystal structures, conformational behavior

NMR spectroscopy, X-ray diffraction analysis, and quantum chemical calculations were used for conformational behavior study of partially alkylated thiacalix[4]arenes bearing methyl (1), ethyl (2), or propyl (3) groups at the lower rim. The conformational properties are governed by two basic effects: (i) stabilization by intramolecular hydrogen bonds, and (ii) sterical requirements of the alkoxy groups at the lower rim. While the monosubstituted derivatives 1a and 3a adopt the cone conformation in solution, distally disubstituted compounds 1b, 1'b, 2b, 2'b, 3b, and 3'b exhibit several interesting conformational features. They prefer pinched cone conformation in solution, and, except for 3'b, they form also 1,2-alternate conformation, which is flexible and undergoes rather fast transition between two identical structures. The crystal structures of the compounds 1b, 2b, 2'b, and 3b revealed yet quite rare 1,2-alternate conformation forming molecular channels held together by pi-pi interactions. Different channels-with hexagonal symmetry, 0.26 nm wide-are formed in the crystal structure of the pinched cone conformation of 3b. An uncommon hydrogen bonding pattern was found in dimethoxy and dipropoxy derivatives 1'b and 3'b that adopt distorted cone conformations in crystal. Trialkoxy-substituted compounds 1c and 3c adopt the partial cone conformation in solution. A higher mobility of methyl derivative 1c enables also existence of the cone conformer.     

Novel bicyclic nucleoside analogue (1S,5S,6S)-6- hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane: synthesis and incorporation into oligodeoxynucleotides

The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane [2'-deoxy-1'-C,4'-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4'-endo furanose conformation, was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 5. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides using standard methods, and thermal denaturation studies showed moderate decreases in duplex stabilities of -2.1 and -1.5 degrees C per modification toward complementary DNA and RNA, respectively.     

7-Functionalized 7-deazapurine ribonucleosides related to 2-aminoadenosine, guanosine, and xanthosine: glycosylation of pyrrolo[2,3-d]pyrimidines with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose

[reaction: see text] The Silyl-Hilbert-Johnson reaction as well as the nucleobase-anion glycosylation of a series of 7-deazapurines has been investigated, and the 7-functionalized 7-deazapurine ribonucleosides were prepared. Glycosylation of the 7-halogenated 6-chloro-2-pivaloylamino-7-deazapurines 9b-d with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (5) gave the beta-D-nucleosides 11b-d (73-75% yield), which were transformed to a number of novel 7-halogenated 7-deazapurine ribonucleosides (2b-d, 3b-d, and 4b-d) related to guanosine, 2-aminoadenosine, and xanthosine. 7-Alkynyl derivatives (2e-i, 3e-h, or 4g) have been prepared from the corresponding 7-iodonucleosides 2d, 3d, or 4d employing the palladium-catalyzed Sonogashira cross-coupling reaction. The 7-halogenated 2-amino-7-deazapurine ribonucleosides with a reactive 6-chloro substituent (18b-d) were synthesized in an alternative way using nucleobase-anion glycosylation performed on the 7-halogenated 2-amino-6-chloro-7-deazapurines 13b-d with 5-O-[(1,1-dimethylethyl)dimethylsilyl]-2,3-O-(1-methylethylidene)-alpha-D-ribofuranosyl chloride (17). Compounds 18b-d have been converted to the nucleosides 19b-d carrying reactive substituents in the pyrimidine moiety. Conformational analysis of selected nucleosides on the basis of proton coupling constants and using the program PSEUROT showed that these ribonucleosides exist in a preferred S conformation in solution.     

Development and conformational analysis of a pseudoproline-containing turn mimic

The liquid-phase synthesis and the conformational analysis of a small library of fully protected tetramers containing L-pyroglutamic acid (L-pGlu), (4S,5R)-4-methyl-5-carboxybenzyloxazolidin-2-one (L-Oxd), or (4R,5S)-4-methyl-5-carboxybenzyloxazolidin-2-one (D-Oxd) as residue i + 1 are reported to test the tendency of these oligomers to assume a -hairpin conformation. The most promising molecule is Boc-L-Val-D-Oxd-Gly-L-Ala-OBn, which assumes a preferential -turn conformation in CDCl3, as shown by IR and 1H NMR analysis. These findings have been confirmed by DFT calculations, which provide an interpretation for the available experimental data and agree with the reported observations.     

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.     

Nuclear magnetic resonance spectra of psychotherapeutic agents. V*—conformational analysis of 1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-ones

The conformational analysis of biologically active lofendazam (7-chloro-5-phenyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one) is carried out by means of lanthanide shift reagent assisted ¹H NMR spectroscopy: the lanthanide induced shift computer simulation suggests that in deuteriochloroform the heterocyclic ring of lofendazam assumes a cycloheptene-like chair conformation, where 1-N moves away from trigonal stereochemistry to a very flattened pyramidal structure. At room temperature the conformational equilibrium is markedly shifted (85%) towards the conformer showing pseudoaxial H-1 and 5-Ph. The remarkable influence of steric requirements in controlling conformation, and the importance of 3- and/or 4-methyl groups in hindering the ring inversion at room temperature, have been verified by conformational analysis of suitable analogous 1,3,4,5-tetraydro-2H-1,5-benzodiazepin-2-ones.     

Addition of difluorocarbene to 3',4'-unsaturated nucleosides: synthesis of 2'-deoxy analogues with a 2-oxabicyclo[3.1.0]hexane framework

Treatment of protected 2'-deoxy-3',4'-unsaturated nucleosides derived from adenosine and uridine with difluorocarbene [generated from bis(trifluoromethyl)mercury and sodium iodide] gave fused-ring 2,2-difluorocyclopropane compounds. Stereoselective alpha-face addition to the dihydrofuran ring resulted from hindrance by the protected beta-anomeric nucleobases. A protected uracil compound was converted smoothly into the cytosine derivative via a 4-(1,2,4-triazol-1-yl) intermediate. Removal of the protecting groups gave new difluorocyclopropane-fused nucleoside analogues. The solid-state conformation of the nearly planar furanosyl ring in the uracil compound had a shallow 2E pucker, and a more pronounced 1E conformation was present in the furanosyl ring of the cytosine derivative.     

Synthesis, conformation, and complexation of novel 25,26,27,28- tetrahydroxy-5,11:17,23-bis[[2,2'-thioxydi(o-phenylene)dithioxy]- diphenylthio]calix[4]arene

Calix[4]arenes 1a,b having an electron-donating group, i.e., OH and OMe, at the lower rim reacted with thianthrene cation radical perchlorate in CH3CN at room temperature to give the corresponding thianthrenium perchlorates 3a,b in excellent yields. Treatment of 3a,b comprising a mixture of conformational isomers with NaSH.xH2O in DMF at reflux afforded the sulfur-containing cyclized compounds 4a,b, respectively. Compound 4a having a cone conformation consisted of two conformational isomers in a ratio of 0.077:1. Temperature-dependent 1H NMR study in DMF showed that conformational isomerization between the two isomers occurred with energy barriers of 14.97 and 14.10 kcal/mol at 100 and 110 degrees C, respectively. The Jobs plot of 4a against Ag+ picrate indicated that compound 4a strongly produced a 1:2 complex with Ag+ ions. Molecular mechanics calculations indicated that the conformation of the energy-minimized 4a-2Ag+ picrate complex had two crushed pyramidal geometries made up of Ag+ ion and four sulfur atoms, i.e., S1, S2, S4, S5 and S6, S7, S9, S10, respectively.     

Crystal and molecular structures of carbon-bridged pyrimidine cyclonucleosides. Substrate analogs of ribonuclease A

The crystal and molecular structures of carbon-bridged 6,5'-cyclo-5'-deoxy-4-thiouridine (6,5'-Cs4U), 6,5'-cyclo-5'-deoxy-2',3'-O-isopropylideneuridine (6,5'-CiU) and 6,6'-cyclo-5',6'-dideoxy-allofuranosyluracil (6,6'-CU) have been determined by X-ray diffraction. The molecular conformations of 6,5'-Cs4U and 6,5'-CiU are very similar; the conformation about the glycosidic bond is anti (low region), the torsion angle O(4')-C(1')-N(1)-C(2) being -150.0 degrees for 6,5'-Cs4U and -145.5 degrees for 6,5'-CiU, and the sugar puckering being both O(4')-exo. On the other hand, 6,6'-CU takes the glycosidic torsion angle of -116.9(4) degrees (middle anti region) and the sugar conformation of C(4')-endo. The cyclization causes little alteration in the geometry of the base moiety. 6,5'-Cs4U and 6,5'-CiU exhibit the similar base-base interactions between adjacent molecules, although their molecular packings are quite different; the 4-thiouracil or uracil moiety interacts with adjacent base moieties through hydrogen bonding and stacking interactions. In 6,6'-CU, cyclonucleosides were connected by hydrogen bondings between the hydroxyl and sugar ring oxygen atoms and between the hydroxyl groups and the base nitrogen and oxygen atoms. As the 2',3'-cyclic phosphates of these carbon-bridged cyclonucleosides are hydrolyzed by ribonuclease A, it is suggested that the conformers found in these cyclonucleosides are recognized by the enzyme.     

Practical synthesis of 2'-deoxy-4'-thioribonucleosides: substrates for the synthesis of 4'-thioDNA

We report herein a practical synthesis of 4'-thiothymidine (15) and appropriately protected 2'-deoxy-4'-thiocytidine (16), -thioadenosine (27), and -thioguanosine (29) derivatives, substrates for the synthesis of 4'-thioDNA, from the corresponding 4'-thioribonucleosides. 2'-deoxy-4'-thiopyrimidine nucleosides were synthesized using a radical reaction of the corresponding 2'-alpha-bromo derivatives, which were prepared via 2,2'-O-anhydro derivatives. 2'-deoxy-4'-thiopurine nucleosides were synthesized using the same radical reaction of the corresponding 2'-beta-bromo derivatives.     

Studies on antitumor agents. IX. Synthesis of 3'-O-benzyl-2'-deoxy-5-trifluoromethyluridine

A practical synthesis of 3'-O-benzyl-2'-deoxy-5-trifluoromethyluridine (1), a candidate antitumor agent for clinical testing, was developed from 2'-deoxy-5-iodouridine (3). Benzylation of 2'-deoxy-5-iodo-5'-O-trityluridine (14) with benzyl bromide and sodium hydride in tetrahydrofuran gave the 3'-O-derivative (16). Benzoylation of 16 afforded the N3-benzoyl derivative (17). Coupling of 17 with trifluoromethylcopper, prepared from bromotrifluoromethane and copper powder in the presence of 4-dimethylaminopyridine, gave the 5-trifluoromethyl derivative (19) minimally contaminated with the 5-pentafluoroethyl compound. Deprotection of 19 furnished 1.     

Synthesis and NMR spectral studies of some 2,6-diarylpiperidin-4-one O-benzyloximes

Variously substituted 2,6-diarylpiperidin-4-one O-benzyloximes were synthesized by the direct condensation of the corresponding 2,6-diarylpiperidin-4-ones with O-benzylhydroxylamine hydrochloride. All the synthesized compounds are characterized by IR, Mass and NMR spectral studies. NMR spectral assignments are made unambiguously by their one-dimensional (1H NMR and 13C NMR) and two-dimensional (1H-1H COSY, NOESY, HSQC and HMBC) NMR spectra. All the synthesized compounds are resulted as single isomer, i.e., exclusively E isomer (9-14). The conformational preference of 2,6-diarylpiperidin-4-one oxime ethers with and without alkyl substituents at C-3 and C-5 has also been discussed using the spectral studies. The observed chemical shifts and coupling constants suggest that compounds 8-13 adopt normal chair conformation with equatorial orientation of all the substituents while compound 14 contributes significant boat conformation along with the predominant chair conformation in solution. The effect of oximination on ring carbons, their associated protons, alkyl substituents and ipso carbons are studied. Every proton in the piperidone ring of the oxime ether is observed as distinct signal due to oximination. The order of chemical shift magnitude in compound 8 is H-2a>H-6a>H-5e>H-3e>H-3a>H-5a. For 9-12, the order is H-6a>H-5e>H-2a>H-3a>H-5a, for 13, H-6a>H-2a>H-5e>H-3a>H-5a and for 14, the order is H-2a>H-6a>H-5e>H-3a>H-5a while the 13C chemical shift magnitude for 8-14 due to oximination is C-2>C-6>C-3>C-5.     

Synthesis of new 3'-, 5-, and N(4)-modified 2'-O-methylcytidine libraries on solid support

A versatile solid phase combinatorial approach was developed and utilized for the rapid synthesis of new 2'-O-methylcytidine nucleoside libraries 1-7 containing 672 compounds with 3'-deoxy-3'-C-methyl, 3'-deoxy-3'-C-hydroxymethyl, and 5-alkyl/alkynyl modifications. The modified uridine scaffolds 8-10, 23-25, and 31 were loaded onto the 4-methoxytrityl chloride (MMT-Cl) polystyrene resin through the hydroxyl groups at the 5'-position as well as on the substituents at the 3'- and 5-positions. The scaffolds loaded on the resin were orthogonally protected by MMT group on the resin itself and TBDMS or acetyl protecting groups. The 4-position of the uridine derivatives was activated by 2,4,6-triisopropyl benzene sulfonyl chloride for further derivatization. The resins 14-16, 28-30, and 32 loaded with the corresponding activated scaffolds were reacted with the selected and validated amino building blocks in the 96 well format on the semiautomated synthesizer. The high-quality 2'-O-methylcytidine libraries 1-7 were thus generated and characterized by liquid chromatography-mass spectrometry (LC-MS) analysis with 63-99% successful rates.     

Proline-mediated formation of novel chroman-4-one tetrahydropyrimidines

Novel tricyclic N-benzylated chroman-4-one tetrahydropyrimidine derivatives have been prepared through a multi-component reaction between various 2-substituted chroman-4-one derivatives, N-methylenebenzylamine and a catalytic amount of proline under mild reaction conditions. The tricyclic structure of 1a was determined by NMR spectroscopy and confirmed by X-ray crystallography. An additional product, 2a, was isolated from the reaction mixture and its structure and conformation were determined by a combination of theoretical (Monte Carlo conformational search) and NMR-based (NOE and ³J_HH couplings) conformational analysis. The NMR analysis revealed one preferred geometry for 1a and 2a in CHCl₃ solution.     

Macromolecular chirality induction on optically inactive poly(4-carboxyphenyl isocyanide) with chiral amines: a dynamic conformational transition of poly(phenyl isocyanide) derivatives

Optically active polyisocyanides (poly(iminomethylenes)) have been prepared with much interest in developing new functional materials. Polyisocyanides have been considered to have a stable 4(1) helical conformation even in solution when they have a bulky side group. However, the conformational characteristics of poly(phenyl isocyanide) (PPI) derivatives are still under debate. We now report that an optically inactive PPI derivative, poly(4-carboxyphenyl isocyanide) (poly-1), shows optical activity in the polymer backbone induced by external, chiral stimuli through acid-base interactions under thermodynamic control and exhibits induced circular dichroism (ICD) in the UV-visible region in DMSO. The ICD intensities of the poly-1-chiral amine complexes in DMSO gradually increased with time, and, in one case, the value reached 3 times that of the original value after 2 months at 30 degrees C. The conformational changes also occurred very slowly for poly-1 alone and its ethyl ester with time on the basis of (1)H NMR spectroscopic analysis. These results indicate that PPIs bearing a less bulky substituent may not have a 4(1) helical conformation but have a different type of prochiral conformation, for instance, an s-trans (zigzag) structure which may transform to a dynamic, one-handed helical conformation when the PPIs have a functional group capable of interacting with chiral compounds. The mechanism of helicity induction on poly-1 through a dynamic conformational transition is discussed on the basis of the above results together with molecular dynamic simulation results for PPI.