Hybridon, Inc.

Avecia Biotechnology, Inc.

Worcester Foundation for Biomedical Research

Jin-Yan Tang

Sudhir Agrawal

Jimmy X Tang

Warren E Schwartz

Zhaoda Zhang

Jose Gonzalez

Nandkumar Bongle

Nandkumar Bhongle

Guangrong Zhang

Zhoada Zhang

Yongxin Han

Saroj K Roy

Saroj Roy

The invention provides improved processes for oligonucleotide synthesis utilizing arenes as solvents for detritylation of oligonucleotides.

Detritylation solvents for nucleic acid synthesis

The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleotide linkages of oligonucleotides. The invention provides a process to synthesize new sulfur transfer reagents and a process for their use in sulfurizing oligonucleotides. The sulfur transfer reagents according to the invention are inexpensive to make, stable in storage and in solution, and highly efficient in sulfurization.

Sulfurizing reagent: 3-aryl-1,2,4-dithiazoline-5-ones

The invention provides passivated organic polymer supports, processes for their preparation and processes for their use in oligonucleotide synthesis that allow for highly efficient solid phase synthesis of oligonucleotides.

Polymer supports for nucleic acid synthesis

The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleoside linkages of oligonucleotides. The invention provides new sulfur transfer reagents and processes for their use in sulfurizing oligonucleotides. The sulfur transfer reagents according to the invention are inexpensive to make, stable in storage and highly efficient in sulfurization.

Sulfur transfer reagents for oligonucleotide synthesis

The invention provides novel bifunctional phosphitylating reagents and their application in in situ preparation of 5&prime;-protected nucleoside phosphoramidites and synthesis of oligonucleotides. Bifunctional phosphitylating reagents according to the invention react quickly with nucleosides under neutral or weakly basic conditions, without an additional activation step. In addition, the bifunctional phosphitylating reagents according to the invention generate chemoselectively the corresponding nucleoside phosphoramidites in situ, without need to purify the nucleoside phosphoramidites before using them in oligonucleotide synthesis. Finally, the bifunctional phosphitylating reagents according to the invention are relatively stable and easy to handle.

Preparation of nucleoside phosphoramidites and oligonucleotide synthesis

The present invention comprises an improved method of synthesizing oligonucleotides. The method comprises employing dinucleotides (or &ldquo;dimer blocks&rdquo;) as the basic synthetic unit building block. The method results in extremely high purity oligonucleotides in which the N&minus;1 content is very low, generally less than 1-2% of the full length, N, oligonucleotide. We have found that synthesis using dinucleotide phosphorothioates results in oligonucleotides having very little phosphodiester content. Furthermore, we have found that the amount of dimer required in each coupling step can be less than about 6 and is preferably about 2 equivalents. Synthesis of oligonucleotides according to the dimer block approach described herein can also be conducted without the capping step that has heretofore been deemed necessary after each coupling.

Extremely high purity oligonucleotides and methods of synthesizing them using dimer blocks

In our research, two commercially available compounds, 3-amino-1,2,4-dithiazole-5-thione (1) and xanthane hydride (2), and their derivatives 3-6 are found to be potential sulfur-transfer reagents. The efficiency and optimization of these new sulfur-transfer reagents were investigated by solid-phase syntheses of dinudeotide and oligonucleotide phosphorothioates. The results show that both compounds 1 and 2 are highly efficient sulfurizing reagents, and better than 99% sulfur transfer efficiency can be achieved at each step. In contrast to Beaucage reagent, these novel sulfur-transfer reagents are very stable in various solvents, and are available in bulk quantities at low cost. Due to these advantages, compounds 1 and 2 can be considered alternatives to Beaucage reagent, especially in large-scale preparation of oligonucleotide phosphorothioates. Furthermore, compounds 1 and 2 were modified to enhance their solubility in acetonitrile. Three types of structure, as described as Formulae I, II, and III, are considered as potential efficient sulfur-transfer reagents based on our results.

The present invention comprises an improved method of synthesizing oligonucleotides. The method comprises employing dinucleotides (or 'dimer blocks') as the basic synthetic unit building block. The method results in extremely high purity oligonucleotides in which the N-1 content is very low, generally less than 1-2% of the full length, N, oligonucleotide. We have found that synthesis using dinucleotide phosphorothioates results in oligonucleotides having very little phosphodiester content. Furthermore, we have found that the amount of dimer required in each coupling step can be less than about 6 and is preferably about 2 equivalents. Synthesis of oligonucleotides according to the dimer block approach described herein can also be conducted without the capping step that has heretofore been deemed necessary after each coupling.

Extremely high purity oligonucleotides and methods of synthesizing them

The present invention provides an improved method of synthesizing 2'-O--R substituted pyrimidine mononucleosides. The method comprises reacting an anhydropyrimidine with magnesium alkoxide in the corresponding alcohol at elevated temperatures to directly produce the 2'-O--R substituted pyrimidine nucleoside product. The method advantageously eliminates several steps from prior art methods, thereby reducing the time and cost of synthesis and increasing the yield of final product.

Method for synthesizing 2'-O-substituted pyrimidine nucleosides

Passivated polymer supports for nucleic acid synthesis

The invention relates to synthetic oligonucleotides and more particularly to the determination of nucleotide sequences of synthetic oligonucleotides having non-phosphodiester internucleotide linkages. The invention provides a method for sequencing such modified synthetic oligonucleotides.

Method for sequencing synthetic oligonucleotides containing

Disclosed are methods of producing a synthetic oligonucleotide of selected nucleotide sequence which is internally functionalized at at least one selected location with an aminoalkyl-phosphoramidate and labelled with a nonradioactive material. Also disclosed are oligonucleotides produced by this method.

Linear aminoalkylphosphoramidate oligonucleotide derivatives

New methods of loading solid supports for oligonucleotide synthesis are presented. The new methods comprise coupling a succinylated nucleoside to a solid support using diisopropylcarbodiimide (DIC) as an activator and N-hydroxybenzotriazole (HOBT) as an acid catalyst. DIC is substantially cheaper than the currently used activating agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). Furthermore, in the preferred embodiment, when it is used in combination with HOBT, coupling is more efficient, requiring less nucleoside to achieve the same loading densities as in the prior art. The loading process is faster than prior art methods and the overall cost savings of about 43% are realized.

Method for loading solid supports for nucleic acid synthesis

Disclosed are methods of producing a synthetic oligonucleotide of selected nucleotide sequence which is internally functionalized at least one selected location with an aminoalkylphosphotriester, and labelled with a nonradioactive material. Also disclosed are oligonucleotides produced by this method.

Aminoalkylphosphotriester oligonucleotide derivatives

Disclosed are methods of producing a synthetic oligonucleotide of selected nucleotide sequence which is internally functionalized at at least one selected location with an aminoalkylphosphorothioamidate and labelled with a nonradioactive material. Also disclosed are oligonucleotides produced by this method.

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Shrewsbury, MA, United States of America

Jin-Yan Tang