4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new orthoester-type protecting group for the 2'-hydroxyl function in the chemical synthesis of RNA.

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2'-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2'-hydroxyl group of appropriately protected nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety of this protecting group with an amine resulted in formation of a new amide moiety that is more stable to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in organic solvents. In this article, we also describe the stability of not only the original and modified protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under deprotection conditions.

Synthesis of N-labeled peptidyl AMP.

This paper deals with the synthesis of a new type of N-labeled peptidyl AMP, which would be used as a good substrate for analysis of the peptidyl transfer reaction on ribosome and for co-crystallization with ribosome. 4-(Dimethylamino)azobenzene-4'-sulfonyl (Dabsyl) was selected as the labeling group. (N-Dabsylglycyl)-L-leucyl AMP was synthesized from glycyl-L-leucine via a three-step procedure.

A new coupling method for the synthesis of oligodeoxyribonucleotides based on protection by protonation of nucleoside bases.

We developed a conceptually new strategy for the coupling method in the phosphoramidite method without protection of nucleoside bases. This strategy, called "proton-block method", significantly reduced side reactions compared with the previous methods.

Synthesis and properties of a new oligonucleotide analogue containing an internucleotide squaryl amide linkage.

We report here the synthesis and properties of a new oligonucleotide analogue (5'-CGCATsqTAGCC-3') containing a squaryl group in place of the fifth phosphate group from the 5'-terminus. The modified thymidine dimer building unit was synthesized by use of 5'-amino-5'-deoxythymidine and a 3'-amino-3'-deoxythymidine derivative. This building unit was phosphitylated and incorporated into the oligonucleotide by the phosphoramidite method. The hybridization ability of this modified oligomer with the complementary oligonucleotide was studied in detail by using Tm experiments and CD spectroscopy as well as computer simulations. These results suggest that this modified duplex has a unique bend conformation.

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

Chemical synthesis of hydroxymethylphosphonate alpha-DNA.

In this paper, we report the synthesis of hydroxymethylphosphonate alpha-DNAs and related compounds by use of the H-phosphonate method. These modified alpha-DNAs were designed to improve the inherent poor solubility of well-known methylphosphonate alpha-DNAs by introduction of a more hydrophilic hydroxymethylphosphonate function. The hybridization ability of hydroxymethylphosphonate alpha-DNAs was studied. We also report a novel strategy for the synthesis of alpha-thymidine by use of C1'-epimerization. The details of the neighboring effect of various 5'- and 3'-hydroxyl protective groups such as carbamoyl groups on the beta-->alpha conversion will be described.

Solid-phase synthesis of deoxyribonucleotides having a cap structure analog.

In this study, the solid-phase synthesis of oligodeoxyribonucleotides having a guanosine pyrophosphate cap structure (Gpp-) was achieved by using a new guanosine monophosphate unit having the DMTr group capable of estimation of coupling efficiency of the pyrophosphate bond formation. Since 7-methylguanosine base was unstable under basic conditions, Gpp-capped DNA oligomers were synthesized by using a new linker having a silanediyl bond, which allowed to release the DNA chain from the solid support by treatment with fluoride anion under neutral conditions.

Synthesis and properties of oligonucleotides having a phosphorus chiral center by incorporation of conformationally rigid 5'-cyclouridylic acid derivatives.

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um as a chiral center at the phosphate group with the S/N junction where c3 refers to a propylene bridge linked between the uracil 5-position and 5'-phosphate group of pUm. The extensive H1 NMR analysis of Umpc3Um suggests that the 5'-upstream Um has predominantly a C2'-endo conformation and the pc3Um moiety exists almost exclusively in a C3'-endo conformation. The absolute configuration of the diastereomers Umpc3Um(fast) (8a) and Umpc3Um(slow) (8b) was determined by CD spectroscopy as well as computer simulations. The oligonucleotides U4[Umpc3Um(fast)]U4 (13a) and U4[Umpc3Um(slow)]U4 (13b) incorporating 8a and 8b were synthesized by use of the phosphoramidite building blocks 11a and 11b, respectively. The Tm experiments of the duplexes formed between these modified oligomers and the complementary oligomers imply that the modified oligomer 13a having Umpc3Um(fast) has the Sp configuration at the chiral phosphoryl group.

Synthesis and properties of oligodeoxynucleotides incorporating a conformationally rigid uridine unit having a cyclic structure at the 5'-terminal site.

A 2'-O-methyluridylic acid derivative 3 having a cyclic structure linked between the 5-position of the uracil residue and the 5'-phosphate group was synthesized. The NMR analysis suggests that this cyclouridylic acid derivative has exclusively the C3'-endo conformation that is in favor of duplex formation with RNA. Two oligonucleotides ¿pc3Um(pT)(9) and pc3Um(pU)(9) incorporating this cyclouridylic acid unit at the 5'-terminal site were synthesized by using the fully protected cyclouridylic acid 3'-phosphoramidite derivative 11 in the solid-phase synthesis. To examine the actual effect of this cyclic structure on the thermal stability of duplexes between the modified oligonucleotides and their complementary oligonucleotides, two oligonucleotides ¿pUm(pT)(9) and pUm(pU)(9) having an acyclic structure were also synthesized. As the complementary oligonucleotides, dA(pdA)(9) and A(pA)(9) were used for T(m) experiments with these 5'-terminal modified oligonucleotides. The T(m) values of all the possible duplexes were measured. These results clearly show that the duplex of pc3Um(pT)(9)-A(pA)(9) has a higher T(m) value by 5.5 degrees C than that of A(pA)(9)-T(pT)(9). This is rather significant compared with all other cases. Moreover, the T(m) value of pc3Um(pT)(9)-A(pA)(9) is 4.5 degrees C higher than that of pUm(pT)(9)-A(pA)(9). This result suggests that the cyclic structure can considerably contribute to stabilization of the duplex only in the case of the modified oligomer (DNA) and decaadenylate (RNA).

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine.

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.

Synthesis of oligonucleotide having a bent structure by incorporation of an interresidually cyclized uridylyl (3'-5')uridine unit.

The chemical synthesis and conformational properties of interresidually cyclized uridylyl(3'-5')uridine derivatives were studied in order to introduce a stable turn structure into oligonucleotides. These cyclized molecules were analogs of uridylyl(3'-5')5-[methylamino(methyl)]-uridine which is the component of the U turn structure of tRNAarg E. coli. The conformational properties of these cyclic dinucleoside monophosphates were studied using NMR and CD spectroscopy with the aid of molecular mechanics and molecular dynamics simulations. These experiments indicated that the turn conformation could be stabilized by introducing a cyclic structure as expected. On the basis of these results, the chemical synthesis of phosphoramidite units of these cyclic dinucleoside monophosphate derivatives were studied to construct oligonucleotides having a stable bent structure.

Binding specificities of the mismatch binding protein, MutS, to oligonucleotides containing modified bases.

We have studied the effects of DNA mismatch repair on mutagenesis induced by nucleoside analogs. Among them, the mutagenic action of 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP) showed high susceptibility to the mismatch repair system, while mutagenesis by N4-aminocytidine and N4-hydroxycytidine was only weakly affected. 2-Aminopurine mutagenesis showed intermediate susceptibility. MutS protein specifically bound to an oligonucleotide duplex containing a dP-dG pair, while the dP-dA pair was bound only weakly. The binding to the dP-dG pair was as strong as binding to a dA-dC mismatch. These specific binding properties can explain the effective avoidance of dP-induced mutagenesis by the mismatch repair system. We have also studied the effects of the repair system on mutagenesis induced by methylating and ethylating agents.