Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1992-07-24
1996-12-31
Robinson, Douglas W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 2533, 536 2534, C07H 2100
Patent
active
055895867
DESCRIPTION:
BRIEF SUMMARY
The present invention relates generally to oligonucleotide synthesis and, particularly, to a new and improved way of attaching the first nucleoside to the solid support that enables oligonucleotides to be more easily and effectively synthesized, deprotected and purified.
Oligonucleotides are polymers built up by polycondensation of ribonucleoside (RNA) or deoxyribonucleoside (DNA) phosphates. The availability of synthetic oligonucleotides has revolutionized molecular biology research. Some of the most important applications of synthetic oligonucleotides are as primers for DNA-sequencing, as primers in the polymerase chain reaction (PCR), as probes for the identification and/or isolation of genes, as building blocks for gene synthesis, and as potential anti-viral compounds in the form of anti-sense oligonucleotides (Ref. 1-3).
Oligonucleotides can be assembled by repetitive addition of nucleotide monomers using solid-phase methods (Ref. 4). Since the introduction of solid-phase synthesis by Merrifield (Ref. 5), the following requirements have been worked out: (1) The solid support must be insoluble and preferably unswellable in the solvent used. (2) Functional groups on the solid support must allow covalent binding of the first nucleoside in a reproducible manner. (3) The solid support must be chemically inert to all reagents used during synthesis and deprotection.
A number of materials have been tested but the most commonly used today are controlled pore glass beads (CPG), silica, or polystyrene beads (Ref. 4).
BRIEF DESCRIPTION OF THE DRAWINGS
The first 5'-protected nucleoside is attached to the support via an alkylamine spacer through the 3'-hydroxyl function. This is normally done by condensation of protected nucleoside 3'-succinate esters with the support-bound alkylamine (see FIG. 1).
This means that four different supports are used, one for each nucleoside (A,C,G,T). Synthesis of oligonucleotides is today almost exclusively carried out by fully automated instruments, by the so called "Gene Machines".
The steps needed to carry out the addition of one nucleotide monomer to the growing chain on the so-lid support using the phosphoramidite method (Ref. 6-8) can be summarized as described below in what is normally referred to as the synthesis cycle, (see FIG. 2). Other methods differ mainly in the nature of the phosphorous moiety (e.g. Phosphotriester (Ref. 11), H-phosphonate (Ref. 9-10) method).
1. Deprotection of the 5'-hydroxyl group in order to generate the parent hydroxyl compounds. This is normally done by treatment of the support with di- or trichloroacetic acid in an organic solvent (for removal of DMTr).
2. The support is washed in order to remove traces of acid.
3. The 5'-hydroxyl group is reacted with the 3'-phosphoramidite moiety of a properly protected incoming nucleotide (A,C,G or T) in the presence of an activator (e.g. tetrazole) to form a 3'-5'-phosphite triester.
4. Excess reagents are removed by washing with an appropriate solvent.
5. Unreacted 5'-hydroxyl groups are blocked as acetates (capping).
6. The capping reagents are removed by washing.
7. The phosphite triester is then oxidized to the corresponding phosphate triester. This is normally done by the action of aqueous iodine.
8. The oxidation reagents are removed by washing.
This process is repeated until the desired oligonucleotide sequence has been synthesized. The difference between the cycles is the nucleotide monomer used for coupling in step 3.
The efficiency of this process is very high. Often, coupling efficiencies above 99% can be obtained. An inherent problem though associated with any polymer supported, repetitive synthesis is the stability of the growing oligonucleotide chain to the various reagents that are used during synthesis. One particularly troublesome problem is that of acid-catalysed depurination of deoxyadenosine (Ref. 12), and to a lesser extent deoxyguanosine units, during removal of 5'-protecting groups (detritylation). In this process the bond between the heterocyclic base and the deoxyribose unit is cl
REFERENCES:
Angew, Chem. Int. Ed. Engl., vol. 28, 1989 Joachim W. Engels et al.: "Gene Synthesis", see p. 716-p. 734.
Gait, Oligonucleotide Synthesis. A Practical Approach, IRL Press, Washington, DC, 1984, see p. 46-47, 85-86 and 114-115.
Kumar et al., "Improvements in Oligodeoxyribonucleotide Synthesis: Methyl N,N-Dialkylphosphoramidite Dimer Units of Solid Support Phosphite Methodology,"J. Org. Chem., 49, 4905-4912 (1984).
DeBart et al., "Sugar Modified Olginucleotides: II. Solid Phase Synthesis of Nuclease Resistant .alpha.-Anomeric Uridylates as Potential Antisense Agents," Tett. Lett., 31(25), 3537-3540 (1990).
Hayakawa et al., "The Allylic Protection Method in Solid-Phase Oligonucleotide Synthesis. An Efficient Preparation of Solid-Anchored DNA Oligomers,"J. Am. CHem. Soc., 112, 1691-1696 (1990).
Crane L. Eric
Pharmacia LKB Biotechnology AB
Robinson Douglas W.
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