Rapid synthesis of oligodeoxyribonucleotides: a new solid-phase method.

A method is described that makes use of a new polyamide resin for the rapid synthesis of short oligodeoxyribonucleotides. The method is illustrated by the preparation of two heptadeoxyribonucleotides, d(pT6-C) and d(pC-A-G-T-G-A-T) using a phosphodiester approach. A further development involved use of phenyl isocyanate as an in situ drying agent, which obviated the need for solvent co-evaporation prior tothe internucleotidic coupling steps. Improved fractionation of thymidyl oligonucleotides was obtained by use of a new microparticulate, silica-based anion-exchanger.     

A phosphorbisamidite approach to the synthesis of oligodeoxyribonucleotides and their analogues

Utilities of deoxyribonucleoside 3'-O-phosphorbisdiethylamidites in the synthesis of oligodeoxyribonucleotides and their analogues are described.     

A new linkage for solid phase synthesis of oligodeoxyribonucleotides.

An aryl diisocyanate has been used to attach an appropriately protected 2'-deoxyribonucleoside bearing a free 3'-hydroxyl group, to a long chain alkylamine controlled pore glass support via a urethane moiety, in a simple two step procedure. This obviates the need for the preparation and short column chromatographic purification of the 2'-deoxyribonucleoside-3'-O-succinates required for preparation of the widely used succinyl linked supports. The greater stability of the urethane bond compared to an ester bond led to substantially higher yields of oligodeoxyribonucleotides prepared by the solid phase phosphotriester method. More than twenty oligodeoxyribonucleotides have already been synthesized on the glass support bearing the new linkage.     

A new method for sequence analysis of oligodeoxyribonucleotides.

The structure of an oligodeoxyribonucleotide may be determined by a simple two-dimensional separation on a polyethyleneimine-cellulose thin layer sheet. Chromatography in the first dimension fractionates by chain length a nested set of fragments that are generated by subjecting the oligomer to partial spleen phosphodiesterase degradation and then labelling their non-common ends with 32P using polynucleotide kinase. A subsequent in situ treatment with nuclease Bal 31 produces labelled mononucleotides, and these are identified by chromatography in the second dimension. Since the method does not identify the 3' terminal nucleotide, a convenient procedure involving 3' end labelling followed by enzymatic digestion to monomers has been developed for this purpose. This approach to sequence analysis also has the advantage of permitting assignment of the identity and location of any modified or unusual bases within the oligonucleotide.     

A rapid and convenient synthesis of poly-thymidylic acid by the modified triester approach.

By using anhydrous triethylamine-pyridine to selectively remove the cyanoethyl group from the fully protected oligonucleotide, a substantial improvement has been achieved in yields and the rates of condensation by the modified triester approach from the 5' leads to 3' end. The unreacted oligonucleotide containing the 5'-hydroxy group was removed by treatment with bis (triazolyl)-p-chlorophenyl phosphate after each condensation in situ. These modifications, as exemplified by the synthesis of fully protected T12, T18, T24 and T38 in 80%, 77%, 70% and 50% yields respectively, should allow the ready synthesis of polynucleotides of even longer chain lengths by purely chemical methods.     

A convenient method of isoflavonone synthesis

Reaction of 2-hydroxydeoxybenzoins with bis(dimethylamino)methane in ethanol results in isoflavonones.     

A convenient method of isoflavonone synthesis

Reaction of 2-hydroxydeoxybenzoins with bis(dimethylamino)methane in ethanol results in isoflavonones.     

Filter disc supported oligonucleotide synthesis by the phosphite method

The phosphite approach to oligodeoxynucleotide synthesis has been applied to the filter disc support. The method is exemplified here by the preparation of eight decadeoxynucleotides.     

Use of new phosphonylating and coupling agents in the synthesis of oligodeoxyribonucleotides via the H-phosphonate approach.

New phosphonylating and coupling agents for the synthesis of oligodeoxyribonucleotides via H-phosphonate approach have been developed. Tris(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite, prepared by the reaction of lithium salt of 1,1,1,3,3,3-hexafluoro-2-propoxide with PCl3, reacts with deoxyribonucleosides in the presence of a catalytic amount of triethylamine to produce in the high yield the corresponding deoxyribonucleoside 3'-H-phosphonate units. The use of a new coupling reagent, 1,3-dimethyl-2-chloro-imidazolinium chloride (DMCI) for the internucleotidic H-phosphonate bond formation via the H-phosphonate approach is also discussed in detail.     

Rapid synthesis of oligodeoxyribonucleotides VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support

A new kieselguhr-polydimethylacrylamide support has been used in a continuous flow, column system for solid phase synthesis of oligodeoxyribonucleotides by a phosphotriester procedure. Using only protected mononucleotides a 14-mer, 20-mer and 27-mer were assembled in high repetitive yield using a simple manually operated, bench top apparatus.     

A simple and convenient method for acyclonucleoside synthesis.

Introduction of acyclic chain for synthesis of acyclonucleoside derivatives was achieved in a simple and convenient way. Silylated pyrimidine or purine bases were treated with 1,3-dioxolane, trimethyl chlorosilane and metal iodide, such as KI and NaI, all together at room temperature. By this method, 2-thiopyrimidine derivatives were also obtained in good yield, using 2 molecular equivalents of 1,3-dioxolane.     

A convenient one-step synthesis of L-aminotryptophans and improved synthesis of 5-fluorotryptophan

A one-pot biotransformation for the generation of a series of L-aminotryptophans using a readily prepared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stability upon freeze-drying, and may be stored and used for long periods after its preparation without significant loss of activity.     

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.     

A new approach to the synthesis of efaroxan

Efaroxan was synthetised by cyclisation of the tertiary alcohol 2 which was prepared by the ring opening of the gem-disubstituted epoxide 3 with ortho-metallated fluorobenzene.     

A new convenient method of azide condensation

Summary A new convenient modification of azide condensation is evaluated. The modification includes the use of tetrabutylammonium nitrite and p-toluene sulphonic acid, which are easily dissolvable in organic solvents, for the acidification of the reaction mixture.     

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries.

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.     

A convenient method for the synthesis of oligonucleotide-cationic peptide conjugates

A method was developed for the synthesis of oligonucleotide-cationic peptide conjugates in solution phase by disulfide bond formation. Precipitation was avoided by the easily removable triethylammonium trifluoroacetate (TEATFAc) salt which served at the same time as a buffer of the reaction mixture. The fast and high yielding disulfide bond formation was due to the Npys thio protecting and activating group of Cys. A solution of the free 5-thiol modified oligonucleotide obtained from Poly-Pak purification was used for conjugation.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.