A novel phosphoramidite, N,N-diisopropylamino-2-cyanoethyl-9-anthacenemethyl phosphoramidite 1, was prepared and coupled with the terminal 5'-hydroxyl of support-bound T-10 and the putative phosphite triester intermediate was subsequently reacted with iodine in the presence of either water or a series of primary and secondary amines. The reactivity of I compared to a previously reported benzyl phosphoramidite 2 was also investigated: oxidation of the product of coupling 2 with CPG-T-10-5'OH under aqueous conditions resulted in greater than 30% of the benzyl moiety being retained. In contrast, essentially complete loss of the 9-anthracenemethyl group was observed using I under the same conditions. Oligonucleotides modified with a terminal phosphate monoester, lipophilic, fluorescent or cationic groups were thus prepared. (c) 2005 Elsevier Ltd. All rights reserved.

A novel phosphoramidite; N,N-diisopropylamino-2-cyanoethyl-ortho-methylbenzylphosphoramidite 1, was prepared. The reaction of 1 with DMTrT and subsequent derivatisation of the phosphite triester product under solution-phase, Michaelis–Arbuzov conditions was investigated. Coupling of 1 with the terminal hydroxyl groups of support-bound oligodeoxyribonucleotides and subsequent reaction with an activated disulfide yielded oligonucleotides bearing a terminal, phosphorothiolate-linked, lipophilic moiety. The oligomers were readily purified using RP-HPLC. Silver(I)-mediated cleavage of the phosphorothiolate linkage and desalting of the oligonucleotides were performed readily in one step to yield cleanly the corresponding phosphate monester-terminated oligomers.

The syntheses of 5-(Z)-(3-aminoallyl)- and 5-(3-aminopropyl)-substituted 2′-deoxyuridine and 2′-deoxycytidine are reported. These compounds were derived from the corresponding 5-propargylamine derivative. [Hobbs, F. W. J. Org. Chem. 1989, 52, 3420.] The catalyst we have employed for these reductions is a NiCl2/NaBH4 system, which we have found to be superior to the more conventional palladium-catalysts previously reported with similar compounds.

Amine functionalities were attached to the 5-position of 5-iodo-2'-deoxyuridine via alkyne and alkene linkers using palladium catalysts. The generation of cis-alkene and the alkane were also investigated. Investigations into the feasibility of derivatising the amine function further to the amide were also undertaken.

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O4-methyluridine,O2-methyluridine or 2-pyrimidinone-1-β-d-riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5′-O -(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyl-S-cyanoethyl-4-thiouridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U41, U42 and C25) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme.

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.

Michaelis-Arbusov chemistry was used to prepare O,S-dialkyl 3′-O-nucleosidyl phosphorothiolate triesters in solution and attached to CPG. The support-bound nucleoside was utilised in the synthesis of a pentaribonucleotide that was fully deprotected on the support. Subsequent treatment with a buffered solution of iodine cleaved the RNA from the CPG with concomitant formation of a terminal 2′,3′-cyclic phosphate.

The synthesis of stable bisubstrate analogues of phosphoglycerate kinase (PGK) in which adenylate is attached to non-scissile 1,3-bisphosphoglycerate (1,3-P(2)GA) analogues will be described. The ability of such analogues to form high-affinity complexes with PGK as determined by NMR will be presented.

O-4-Methyluridine, O-2-methyluridine and (4H)uridine phosphoramidites were prepared with 2'-O-silyl protection, The methyl modified pyrimidines were synthesised from uridine via the triazolide (O-4-methyl) or 2,5'-cyclo derivative (O-2-methyl). An improved synthesis of protected 4-thiouridine was also performed.

The 5'-O-monomethoxytrityl-3'-S-(aryldisulfanyl)-3'-deoxythymidines 7 and 8 have been prepared by the reaction of 5'-O-monomethoxytrityl-3'-thiothymidine with the appropriate arenesulfenyl chloride. These disulfides undergo a Michaelis-Arbusov reaction with simple trialkyl phosphites to yield 5'-O-monomethoxytrityl-3'-thiothymidin-3'-yl O,O-dialkyl phosphorothiolates. More interestingly, 3'-deoxy-3'-S- (2,4-dinitrophenylsulfanyl)-5'-O-monomethoxytritylthymidine 8 reacts with a variety of thymidin-5'-yl dialkyl phosphites to give dithymidine phosphorothiolate triesters with the phosphorothiolate group protected with either a methyl or a 2-cyanoethyl group.3'-O-(tert-Butyldimethylsilyl)thymidin-5'-yl triethylammoniumphosphonate 17 is converted into the corresponding bis-(O-trimethylsilyl) phosphite by treatment with bis(trimethylsilyl)trifluoroacetamide. in situ Reaction of this phosphite with disulfide 8 gives, after work-up, the dithymidine phosphorothiolate diester directly. Methylation of compound 17 with methyl chloromethanoate, followed by silylation and subsequent reaction with disulfide 8, gives the methyl-protected dithymidine phosphorothiolate triester.

ALL catalytic RNAs (ribozymes) require or are stimulated by divalent metal ions, but it has been difficult to separate the contribution of these metal ions to formation of the RNA tertiary structure1 from a more direct role in catalysis. The Tetrahymena ribozyme catalyses cleavage of exogenous RNA2,3 or DNA4,5 substrates with an absolute requirement for Mg2+ or Mn2+ (ref. 6). A DNA substrate, in which the bridging 3' oxygen atom at the cleavage site is replaced by sulphur, is cleaved by the ribozyme about 1,000 times more slowly than the corresponding unmodified DNA substrate when Mg2+ is present as the only divalent metal ion. But addition of Mn2+ or Zn2+ to the reaction relieves this negative effect, with the 3' S-P bond being cleaved nearly as fast as the 3' O-P bond. Considering that Mn2+ and Zn2+ coordinate sulphur more strongly than Mg2+ does7,8, these results indicate that the metal ion contributes directly to catalysis by coordination to the 3' oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group. We conclude that the Tetrahymena ribozyme is a metalloenzyme, with mechanistic similarities to several protein enzymes9-12.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.

Efficient procedures are described for the synthesis of dinucleoside phosphorothiolates using either a Michaelis-Arbusov-type reaction or a phosphotriester approach.

Dithymidine-3'-S-phosphorothioate (d(TspT)) has been prepared from a 5'-O-monomethoxytritylthymidine-3'-S-phosphorothioamidite (7) by activation with 5-(p-nitrophenyl)tetrazole in the presence of 3'-O-acetylthymidine. The resulting dinucleoside phosphorothioite is readily oxidised to the corresponding 3'-S-phosphorothioate using either tetrabutylammonium (TBA) periodate or TBA oxone and has been deprotected under standard conditions to yield d(TspT). This dithymidine phosphate analogue is comparatively resistant to hydrolysis by nuclease P1, but the P-S bond is readily cleaved by aqueous solutions of either iodine or silver nitrate. Dithymidine-3'-S-phosphorodithioate (d[Tsp(s)T]) was prepared in an analogous fashion using sulphur to oxidise the intermediate dinucleoside phosphorothioite. Absolute stereochemistry has been assigned to the diastereoisomers of d[Tsp(s)T] by comparing their physical and chemical properties to those of the dinucleoside phosphorothioates.

A 5′-O-monomethoxytritylthymidine-3′-S-thiophosphoramidite (3) has been used to prepare oligodeoxynucleotides containing 3′-thiothymidine on a solid phase support. The intermediate thiophosphites are most efficiently oxidised using tetrabutylammonium periodate.

R. Cosstick and J. S. Vyle, J. Chem. Soc., Chem. Commun., 1988, 992 DOI: 10.1039/C39880000992