Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.     

Aromaticity interplay between quinodimethanes and C(60) in diels-alder reactions: insights from a theoretical study

A theoretical study is performed of the Diels-Alder reactions of various o-quinodimethanes (QDM) with C(60) by the AM1 model and limited ab initio and DFT techniques. All reactions are shown to proceed through a concerted transition state possessing a considerable net aromaticity as evidenced from bond orders and magnetic criteria such as the magnetic susceptibility exhaltations (MSE) and nucleus independent chemical shifts (NICS) and produce different kinds of aromatic stabilized fullerene cycloadducts. Computations show that a strong LUMO-dienophile control of C(60) is realized by the influence of pyramidalization, but its high reactivity over alkene appears to be governed by the global aromaticity on fullerene rather than its strain. The aromatic functionalization occurring in QDM upon cycloaddition drastically increases the reaction rate and exothermicity of all QDM-C(60) reactions as compared to the butadiene-C(60) reaction. In fact, the simultaneously existing aromatic destabilization in fullerene indicates its opposite effect to the resonance stabilization in diene; it is thus fully restricted when the gained aromaticity is transmitted from the nucleophilic QDM to the fullerene electrophile in a push-pull manner. However, the overall aromaticity effect shown by the aromatization as well as the aromaticity of C(60) seems to accelerate these reactions at an increased rate.     

Exchange interactions in some low-dimensional metal dithiolene complexes

Low-dimensional systems are formed by planar metal dithiolene complexes which stack as columnar structures in the solid state. Stronger interactions among units within a chain leads to highly anisotropic magnetic properties. The magnetic effects are a manifestation of exchange interaction,J and can be studied through detailedepr techniques in conjunction with magnetic susceptibility and x-ray crystal structure. A brief review of such studies carried out mostly in our laboratory is presented along with the relevant background materials.     

Structural Basis for Recognition of Guanosine by a Synthetic Tricyclic Cytosine Analogue: Guanidinium G‐Clamp

An oligonucleotide analogue containing a novel heterocyclic analogue, the guanidinium G‐clamp, was designed to allow formation of five H‐bonds to guanosine. The guanidinium group was introduced postsynthetically by treatment of the deprotected oligonucleotide containing a free amino group with a solution of 1H‐pyrazole‐1‐carboxamidine and purified by a combination of size‐exclusion chromatography and reversed‐phase HPLC. A single incorporation of this modification into an oligodeoxynucleotide sequence was found to increase duplex stability by 13° and 16° per modification to RNA and DNA, respectively. Crystals of a self‐complementary decamer sequence containing this modification were grown and diffracted to 1‐Å resolution. The structure was solved by molecular replacement and revealed that the modification forms additional H‐bonds to O(6) and N(7) of guanosine through the amino and imino N‐atoms, respectively. The origins of enhanced duplex stability are also attributed to increased stacking interactions mediated by the phenoxazine moiety of the G‐clamp and formation of H‐bond networks between the positively charged guanidinium group, H₂O molecules, and negatively charged O‐atoms from phosphates on the adjacent strand.     

Thermodynamic and strain effects in the competition between 5-exo-dig and 6-endo-dig cyclizations of vinyl and aryl radicals

Electronic and structural factors controlling the competition between 5-exo-dig and 6-endo-dig cyclizations of sp2-radicals were analyzed using a combination of available experimental data and computation. Although the stereoelectronically favored 5-exo pathways usually has the lower activation energy, formation of a new aromatic ring not only makes the 6-endo process favorable thermodynamically in conjugated systems but also lowers its activation barrier to the extent where the 5-exo/6-endo selectivity is controlled by subtle factors such as the different sensitivity of the two pathways to strain effects in polycyclic systems. In particular, the stronger sensitivity of the 5-exo pathway to strain leads to a crossover in selectivity. The 6-endo cyclization is kinetically favored in smaller (and strained) cycles, whereas the 5-exo cyclization has lower barriers in the larger rings.     

Copper(II) azide complexes of aliphatic and aromatic amine based tridentate ligands: novel structure, spectroscopy, and magnetic properties

Copper(II) azide complexes of three tridentate ligands namely 2,6-(3,5-dimethylpyrazol-1-ylmethyl)pyridine (L), 2,6-(pyrazol-1-ylmethyl)pyridine (L'), and dipropylenetriamine (dpt) yield three kinds of complexes with different azide-binding modes. The ligand L forms two end-on-end (mu-1,3) diazido-bridged binuclear complexes, [CuL(mu-N(3))](2)(ClO(4))(2) (1) and [CuL(mu-N(3))(ClO(4))](2).2CH(3)CN (2), and L' forms a perchlorato-bridged quasi-one-dimensional chain complex, [CuL'(N(3))(ClO(4))](n)() (3) with monodentate azide coordination. The ligation of dipropylenetriamine (dpt) gives a end-on (mu-1,1) diazido-bridged binuclear copper complex [Cu(dpt)(mu-N(3))](2)(ClO(4))(2) (4). The crystal and molecular structures of these complexes have been solved. Variable-temperature EPR results of 1 and 2 are identical and indicate the presence of both ferromagnetic and antiferromagnetic interactions within the dimer, the former dominating at low temperatures and the latter at high temperatures. The unusual temperature-dependent magnetic moment and EPR spectra of this dimer reveal the presence of temperature-dependent population of two triplet states, one being caused by antiferromagnetic and the other by ferromagnetic interaction, the former transforming to the latter on cooling. While the interaction of ground spin doublets of the two metal centers gives rise to a ferromagnetic coupling of J(g) = 90.73 cm(-1), the other coupling of J(e) = -185.64 cm(-1) is suggested to be caused by the interaction between an electron in one metal center and an electron from the azide of the other monomer by excitation of a d-electron to the empty ligand orbital. The ferromagnetic state is energetically favored by 104.39 cm(-1). Compound 3 exhibits axial spectra at room temperature and 77 K, and variable-temperature magnetic susceptibility data indicate that the copper centers form a weakly antiferromagnetic one-dimensional chain with J = -0.11 cm(-1). In the case of 4, the unique presence of two nonidentical dimeric units with different bond lengths and bond angles within the unit cell as inferred by crystal structure is proved by single-crystal EPR spectroscopy.