Barbro Beijer

How glutaminyl-tRNA synthetase selects glutamine

BACKGROUNDnAminoacyl-tRNA synthetases covalently link a specific amino acid to the correct tRNA. The fidelity of this reaction is essential for accurate protein synthesis. Each synthetase has a specific molecular mechanism to distinguish the correct pair of substrates from the pool of amino acids and isologous tRNA molecules. In the case of glutaminyl-tRNA synthetase (GlnRS) the prior binding of tRNA is required for activation of glutamine by ATP. A complete understanding of amino acid specificity in GlnRS requires the determination of the structure of the synthetase with both tRNA and substrates bound.nnnRESULTSnA stable glutaminly-adenylate analog, which inhibits GlnRS with a Ki of 1.32 microM, was synthesized and cocrystallized with GlnRS and tRNA2Gln. The crystal structure of this ternary complex has been refined at 2.4 A resolution and shows the interactions made between glutamine and its binding site.nnnCONCLUSIONSnTo select against glutamic acid or glutamate, both hydrogen atoms of the nitrogen of the glutamine sidechain are recognized. The hydroxyl group of Tyr211 and a water molecule are responsible for this recognition; both are obligate hydrogen-bond acceptors due to a network of interacting sidechains and water molecules. The prior binding of tRNAGln that is required for amino acid activation may result from the terminal nucleotide, A76, packing against and orienting Tyr211, which forms part of the amino acid binding site.

New synthetic routes to protected purine 2′-O-methylriboside-3′-O-phosphoramidites using a novel alkylation procedure

A highly selective alkylation procedure has been developed enabling new synthetic routes to be established for protected purine 2-O-methylriboside-3-O-phosphoramidites; building blocks for the assembly of 2-O-methyloligoribonucleotides. The new procedure avoids the use of the highly toxic and potentially explosive reagent diazomethane and is far superior to the use of silver oxide/methyl iodide. Moreover, the use of highly versatile key intermediates will enable the synthesis of a wide variety of base modified analogues as well as other 2-O-alkylriboside derivatives.

2′-O-alkyloligoribonucleotides Synthesis and Applications in Studying RNA Splicing

Abstract Improved synthetic routes have been established for the preparation of appropriately protected 2′-O-allylribonucleoside-3′-O-phosphoramidites. 2′-O-Allyloligoribonucleotides prove to be superior antisense compounds for investigating RNA processing.

2′-O-(carbamoylmethyl)oligoribonucleotides

Abstract Fully modified oligonucleotides containing 2′- O -cyanomethylribonucleotides were synthesised on automated DNA synthesisers using protected 2′- O -cyanomethylribonucleoside-3′- O -phosphoramidite building blocks. The 2′- O -cyanomethyloligoribonucleotides were post-synthetically converted into 2′- O -carbamoylmethyloligoribonucleotides during standard deprotection conditions with aqueous ammonia. The complete conversion was proven using ion- exchange HPLC and MALDI mass spectrometry of the final oligomer. The 2′- O -carbamoylmethylribonucleotides showed an substantial increase the melting temperature (Tm) of duplexes with complementary RNA relative to the corresponding RNA homoduplex. Consequently these analogues should prove useful for a variety of antisense applications.

Simplified and Cost Effective Syntheses of Fully Protected Phosphoramidite Monomers Suitable for the Assembly of Oligo(2′-O-allylribonucleotides)

Abstract Simplified, high yielding syntheses of suitably protected 2′-O-allylribonucleoside-3′-O-phosphoramidites starting from standard ribonucleosides have been elucidated. Specific 2′-O-allylation is readily achieved using amidine protection of the exocyclic amino groups of adenosine and cytidine and in the case of guanosine the allylation is carried out on an easily prepared intermediate bearing transient protection of the lactam function.

An approach towards thiol mediated labelling in the minor groove of oligonucleotides

Abstract The preparation of an appropriately protected 2′- O -(2-thioethyl)uridine phosphoramidite is reported along with its incorporation into an oligonucleotide and post-synthetic labelling.

Synthesis of modified building blocks containing amino of thiol moieties: application of modified oligodeoxyribonucleotides

Abstract Modified building blocks have been synthesised and used to prepare 5′- amino and 5′-mercapto-oligodeoxyribonucleotides. Subsequent labelling with fluorophores or metal cluster derivatives generates a range of very useful probes.

Novel solid-phase synthesis of branched oligoribonucleotides, including a substrate for the RNA debranching enzyme

An effective new route for synthesizing branched oligoribonucleotides in the solid phase in the 5′ to 3′ direction has been developed. This required the synthesis of reversed monomers, viz. protected nucleoside 5′-phosphoramidites bearing 2′-O-Fpmp and (3′-O-pixy) protecting groups as well as special branch-point monomers, viz. protected nucleoside 5′-phosphoramidites bearing either 2′, 3′-O-dipixyl protection in the case of adenosine, cytidine and uridine, or 2′,3′-O-dilaevulinyl protection in the case of guanosine. These monomers are assembled on commercial synthesizers into branched oligoribonucleotides in high yield, the crude products are readily purified by reversedphase HPLC whilst still partially protected, and the fully deprotected products are conveniently analysed by electrospray mass spectrometry. Moreover, the branched oligoribonucleotides can be recognised and cleaved by a specific 2′–5′ phosphodiesterase present in mammalian cell nuclei. We expect that this will prove valuable for future biochemical and biological studies on the properties of branched RNA molecules and the protein factors and enzymes that interact with branched RNA substrates.

A simple method for the synthesis of 2′-O-alkylguanosine derivatives

Abstract A new synthetic route has been devised for the preparation of 2′- O -alkyl guanosine derivatives. Utilizing a strategy of minimal protection, the alkylation was performed on partly protected guanosine using an alkyl halide and a sterically hindered strong organic base.

Protection of the guanine residue during synthesis of2′-O-alkylguanosine derivatives

Highly selective 2′-O-alkylation of n3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl) nguanosine has been achieved by using an alkyl halide and a sterically nhindered strong organic base, when the 6-O atom is protected with either na 2-nitrophenyl or a tert-butyldiphenylsilyl group prior to the nalkylation. A minimum of chromatography is required, the yields are high nand none of the unwanted isomer is produced. Moreover, the highly nversatile intermediates enable the synthesis of several new n2′-O-alkylguanosine derivatives as well as base-modified nanalogues.