A solid supported reagent for internucleoside H-phosphonate linkage formation

A fast and convenient procedure for synthesis of dinucleoside H-phosphonates is obtained through use of the novel polystyrene supported 5-carboxy-5-methyl-2-oxo-2-chloro-1,3,2-diaoxaphosphorinane reagent. Virtually quantitative H-phosphonate condensations are obtained leading to excellent isolatedyields and with only a simple filtration as the purification procedure. This provides for a convenient and high-yielding procedure that should be suited for solution-phase synthesis of oligonucleotides.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

The diastereoselective synthesis of methyl 5-deoxy-5-(dialkylphosphono)-5-(dialkylphosphorylamido)-2,3-O-isopropylidene-beta-D-ribofuranosides

A convenient method has been developed for the diastereoselective synthesis of methyl 5-deoxy-5-(dialkylphosphono)-5-(dialkylphosphorylamido)-2,3-O-isopropylidene-beta-d-ribofuranosides under mild conditions, namely the reaction of a dialkyl phosphoramidate with a dialkyl phosphite and methyl 2,3-O-isopropylidene-beta-d-ribo-pentodialdo-1,4-furanoside in acetyl chloride in a one-pot procedure.     

Direct regioselective enzymatic acylation of nucleosides: building-blocks for the solution phase synthesis of oligonucleotides

The synthesis of 3'- and 5'-O-levulinyl nucleosidic monomers through enzymatic acylation with acetonoxime levulinate is demonstrated. The acylation process takes place in one-step and use of expensive reagents, such as DMTrCl is avoided. The regioselectivity of the procedure makes it very convenient for acylated monomers required for solution phase synthesis of oligonucleotides.     

Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.     

A short, efficient synthesis of 2'-deoxypseudoisocytidine based on Heck-chemistry

A novel synthesis of 2'-deoxypseudoisocytidine as well as of its phosphoramidite building block for oligonucleotide synthesis is presented. The synthesis is based on Heck-coupling between N-protected pseudoisocytosine and a silyl protected furanoid glycal. With this procedure the corresponding phosphoramidite building block is obtained in 5 steps and an overall yield of 28%.     

The synthesis and use of oligodeoxynucleotides in plasmid DNA sequencing

A convenient procedure for the synthesis and purification of oligonucleotides is described. 16-base long primers synthesised by this method were used to investigate DNA sequencing using plasmid DNA as a template. This allowed the further analysis of the E. coli glt A sequence coding for citrate synthase and enabled determination of the 5'-non-coding regulatory region of the aminoglycoside phosphotransferase gene.     

New efficient synthesis of thymidine cyclic 3',5'-phosphorofluoridate and its sulfur analogue via the phosphoroamidite route

We describe the convenient synthesis of thymidine cyclic 3', 5'-phosphorofluoridate 6, which is superior to that previously reported. Our procedure is based on a sequence of reactions utilizing 3 as the key substrate. Similar sequence of reaction leads to the sulfur analogues of 6 the thymidine cyclic 3',5'-phosphorofluoridothioate 7.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

Low-temperature,high yield synthesis,and convenient isolation of the high-electron-density cluster compound Ta6Br14.8H2O for use in biomacromolecular crystallographic phase determination

Reduction of TaBr(5) with Ga in the presence of KBr in a sealed borosilicate ampule at 400 degrees, followed by aqueous Soxhlet extraction and addition of stannous bromide and hydrobromic acid to the extract, yielded Ta(6)Br(14).8H(2)O in 80-84% yield. The new procedure provides a convenient, low temperature, high yield route to the synthesis of the title compound from inexpensive precursors.     

Direct Regioselective Enzymatic Acylation of Nucleosides: Building-Blocks for the Solution Phase Synthesis of Oligonucleotides

Abstract

The synthesis of 3′- and 5′-O-levulinyl nucleosidic monomers through enzymatic acylation with acetonoxime levulinate is demonstrated. The acylation process takes place in one-step and use of expensive reagents, such as DMTrCl is avoided. The regioselectivity of the procedure makes it very convenient for acylated monomers required for solution phase synthesis of oligonucleotides.     

Regioselective 2′/3′-O-Allylation of Pyrimidine Ribonucleosides Using Phase Transfer Catalysis

Abstract

A short and convenient procedure for regiospecific O-allylation of uridine is reported by employing dibutyltin oxide as a mild base in conjunction with a phase transfer catalyst tetrabutylammonium bromide. The resulting isomeric 2′/3′-O-allyl uridines were separated after conversion into their corresponding 5′-O-DMT derivatives. The 2′-O-allyluridine 3 was then transformed into 2′-O-allylcytidine 7 and both were individually converted into the corresponding β-cyanoethyl phosphoramidite monomers (9 and 10) and a phosphodiester monomer 11, required for oligonucleotide assembly. The utility of 11 is demonstrated by synthesis and characterization of a 2′-O-allyl ribodinucleotide UpU.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores through SPPS-based 'building block' approach

This protocol presents the synthesis and peptide incorporation of environment-sensitive fluorescent amino acids derived from the dimethylamino-phthalimide family. The procedure uses anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol by these authors. In this study, the corresponding fluorescent amino acids can be readily obtained in Fmoc-protected form for convenient use as building blocks in solid phase peptide synthesis (SPPS). The time required to complete the procedure depends on the size and the number of peptides targeted. Alternatively, the chromophores can be incorporated directly after SPPS via on-resin derivatization of peptides, which is an option described in a related protocol by these authors.     

A facile synthesis of DNA oligomers with modified backbones via the phosphoramidite method without nucleoside base protection.

A convenient synthesis of DNA oligomers with modified back bones including phosphorothioates and phosphotriesters has been developed on the basis of the phosphoramidite strategy without nucleoside base protection. In this synthesis, a new, convenient method for the preparation of N-free-5'-O-dimethoxytrityl-2'-deoxyribonucleoside by the direct O-selective dimethoxytritylation of the parent nucleosides has also been disclosed.     

Evaluation of high temperature glycation of proteins and peptides by electrospray ionization mass spectrometry

Recently Boratyński & Roy (Glycoconjugate J., 1998, 15, 131) described a fast and convenient procedure for the synthesis of glycoconjugates. In the present study we used ESI-MS and circular dichroism as tools to analyze non-enzymatic glycation products of proteins and peptides. We discuss influence of reaction conditions on the rate of glycation of lysozyme. We analyze for the first time collision induced dissociation spectra of the obtained peptide conjugates.     

A Short,Efficient Synthesis of 2′-Deoxypseudoisocytidine Based on Heck-Chemistry

Abstract

A novel synthesis of 2′-deoxypseudoisocytidine as well as of its phosphoramidite building block for oligonucleotide synthesis is presented. The synthesis is based on Heck-coupling between N-protected pseudoisocytosine and a silyl protected furanoid glycal. With this procedure the corresponding phosphoramidite building block is obtained in 5 steps and an overall yield of 28%.     

Silica gel: an improved support for the solid-phase phosphotriester synthesis of oligonucleotides.

The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade silica gel (Porasil B) as the solid support. The procedure results in a convenient flow-through system for the synthesis of oligomers where all the reaction steps including the zinc bromide method of detritylation are compatible with the selected support. Deoxyoligonucleotides of 25-30 nucleotides in length can be synthesized in high yields utilising stable phosphotriester intermediates. Ease of handling of the solid support allows convenient synthesis of mixed oligonucleotide sequences.     

New Efficient Synthesis of Thymidine Cyclic 3′, 5′- Phosphorofluoridate and Its Sulfur Analogue via the Phosphoroamidite Route

Abstract

We describe the convenient synthesis of thymidine cyclic 3′, 5′-phosphorofluoridate 6, which is superior to that previously reported. Our procedure is based on a sequence of reactions utilizing 3 as the key substrate. Similar sequence of reaction leads to the sulfur analogues of 6 the thymidine cyclic 3′, 5′-phosphorofluoridothioate 7.     

Solid phase synthesis of a glycopeptide analogue using the acid sensitive 4-methoxybenzhydryl bromide resin

A convenient solid phase synthesis of a Thrombin Receptor Glycopeptide Mimetic analogue namely, 1-O-Methyl-2-N-{1'-(argininocarbonyl)-4'-[(4'-fluoro)-benzylamido]-cyclohexane}-glucosamine using Fmoc/tBu methodology and the 4-Methoxybenzhydryl bromide resin is described. The synthesized analogue was purified by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and was identified by Electron Spray Ionization-Mass Spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR). The synthetic protocol introduced for the first time successfully the acid sensitive 4-Methoxybenzhydryl bromide resin as a scaffold for the synthesis of glycopeptides resulting in high yield reactions. This synthetic procedure could be a general one for the convenient synthesis of such glyco compounds as the method was used for the first time to glycosylate a non peptide mimetic of an important protein sequence, in particular of the thrombin receptor active site S42FLLR46.     

Improved Methods for 3′-O-Succinylation of 2′-Deoxyribo- and Ribonucleosides and Their Covalent Anchoring on Polymer Supports for Oligonucleotide Synthesis

Abstract

A more convenient and efficient method is described for the preparation of 3′-O-succinates of 2′-deoxyribo- and ribonucleosides, in a variety of solvents. Also, a new one-pot procedure has been developed for anchoring these succinates to polymer supports, suitable for solid phase synthesis of oligonucleotides.