Avecia Biotechnology, Inc.

Nanda Dulal Sinha

Satya Kuchimanchi

Gregory Keith Miranda

William Edward Zedalis

Saied Shaikh

A process for the preparation of an oligonucleotide having at least one phosphate internucleotide linkage I provided. The process comprises the steps of:

Oxidation process

A process for the preparation of a poly(alkoxylated) oligonucleotide is provided. The process comprises reacting an oligonucleotide which has been purified by ultrafiltration with a poly(alkoxide) thereby to form a poly(alkoxylated) oligonucleotide. The poly(alkoxyalted) oligonucleotide may be separated from non-poly(alkoxyalted) oligonucleotide by ultrafiltration under denaturing conditions, such as the presence of organic solvents, for example, ethanol; the presence of urea; the addition of chaotropic salts, for example perchlorate and guanidinium salts; the presence of formamide; and the application of heat, for example a temperature of up to about 70&#xb0; C.

Process for the preparation of poly(alkoxylated) oligonucleotides

A process for the synthesis of oligonucleotides using phosphoramidite chemistry is provided. The process employs as activator a 1,1-dioxo-1,2-dihydro-1&#x3bb;-benzo[d]isothiazol-3-one, preferably in the presence of an organic base. The 1,1-dioxo-1,2-dihydro-1&#x3bb;-benzo[d]isothiazol-3-one is represented by the following structural formula: 

Activators for oligonucleotide synthesis

Nitrogen-protecting groups are removed from the exocyclic nucleobase portion of a 2'-O protected nucleotide or 2&#x2032;-halo nucleotide by contacting the nucleotide with an inorganic base. Typical is the removal of t-butylphenoxyacetyl protecting groups from the nucleobase portion of a 2&#x2032;-O protected nucleotide on which the 2&#x2032;-O protecting group is t-butyldimethylsilyl, removal or deprotection being accomplished by contact with a potassium carbonate solution.

Process for the removal of exocyclic base protecting groups

A process for the synthesis of oligonucleotides using phosphoramidite chemistry is provided. The process employs as activator a 1,1-dioxo-1,2-dihydro-1&#x3bb;-benzo[d]isothiazol-3-one, preferably in the presence of an organic base. The 1,1-dioxo-1,2-dihydro-1&#x3bb;-benzo[d]isothiazol-3-one is represented by the following structural formula: wherein p is 0 or an integer from 1 to 4; Xis O or S; R for each occurrence is a substituent, preferably each independently, a halo, a substituted or unsubstituted aliphatic group, &#x2014;NRR, &#x2014;OR, &#x2014;OC(O)R, &#x2014;C(O)OR, or cyano; or two adjacent R groups taken together with the carbon atoms to which they are attached form a six membered saturated or unsaturated ring; Rand Rare each, independently, &#x2014;H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group; and Ris a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group. Preferred organic bases are pyridine, 3-methylpyridine, or N-methylimidazole.

A process for the phosphitylation of an alcohol or thiol with a phosphitylation agent in the presence of an activator is provided. The activator has the formula: wherein p is 0 or an integer from 1 to 4 and R for each occurrence is a substituent. Preferably Xis O and p is 0. The activator is commonly employed as a salt complex with an organic base. Preferred alcohols or thiols include nucleosides and oligonucleotides. The process is particularly suited for the synthesis of phosphoramidites.

Phosphitylation process

A solid support bound polynucleotide, preferably a 3'-O- or 5&#x2032;-O-substituted polynucleotide, is disclosed. The polynucleotides are of formula: wherein Q is a substantially pure polynucleotide, each X is O or S; Y is an inert spacer group; R represents the solid support. Also disclosed is a method of isolating a target biomolecule, preferably a protein or a polynucleotide, from a mixture.

Immobilization of oligonucleotides onto solid supports

The present invention relates to a method of preventing modification of a synthetic oligonucleotide or oligonucleotide analog during removal of at least one &#x3b2;-cyanoethyl protecting group from the oligonucleotide or oligonucleotide analog. The method involves contacting the oligonucleotide or oligonucleotide analog with a basic solution having at least one acrylonitrile scavenger, such as t-butylamine, at a sufficient temperature and for a sufficient period of time to remove at least one &#x3b2;-cyanoethyl protecting group. The present invention also relates to a method of producing a synthetic oligonucleotide or oligonucleotide analog.

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Milford, MA, United States of America

Nanda Dulal Sinha