Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-23
2002-04-30
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S025310, C536S025330, C536S025340, C536S025400, C536S026700, C536S026800
Reexamination Certificate
active
06380378
ABSTRACT:
The present invention relates to a novel nucleotide, nucleotide block and oligonucleotide, and a method for producing the nucleotide block and oligonucleotide. These compounds and production method thereof according to the present invention are useful in synthetic organic chemistry, biochemistry and pharmaceutical industries, for example, as intermediates for the production of oligonucleotides and a method for producing the oligonucleotides.
Hitherto, a solid phase preparation method has been employed for preparation of oligodeoxyribonucleotides and oligoribonucleotides. According to this method, DNA chain or RNA chain is sequentially extended on a solid phase carrier insoluble in various organic solvents. More specifically, in general, using a starting material of a nucleoside whose 3′-hydroxyl group is fixed on an insoluble carrier such as a porous glass, an oligonucleotide chain is extended from 3′ terminal to the direction of 5′ terminal by one base at a time, and according to this method, oligonucleotides of desired sequences can be synthesized (Koester et al, JP-B-62-50479 corresponding to PCT/WO85/00816 and U.S. Pat. No. 4,725,677, and Caruthers et al, JP-B-63-28439 corresponding to U.S. Pat. No. 4,415,732). The above solid phase synthesis method has the merit that the excessively used reagents or solvents can be readily removed at the time of extension of the chain, and each elementary reaction is allowed to proceed by using excess reagents as required, whereby oligodeoxyribonucleotides and oligoribonucleotides having the desired sequence can be produced.
Generally, &bgr;-cyanophosphoramidite developed by Koester et al is used as a nucleotide reagent for extension of DNA chain or RNA chain, and a porous glass is used as a solid phase carrier. See H. Koester et al, “Tetrahedron Lett.”, 52, 5843 (1983) and PCT/WO97/42202.
Furthermore, as the similar nucleotide reagents, there are also known phosphoramidite compounds of the following formula (I-1) where R
2′
and R
3′
are both hydrogen atom and Y is a dialkylamino group. See H. Koester et al, “Tetrahedron Lett.”, 52, 5843 (1983) supra and PCT WO97/42202 supra.
When the above phosphoramidite compounds are used as intermediates for DNA oligomers, generally, a 2-cyanoethoxydialkylaminophosphine derivative is reacted with a 5′-O- and base-protected nucleoside to make a DNA synthesis reagent.
According to this method, starting materials for chemical synthesis of DNA can be stably obtained, but in order to obtain the desired products in a high purity, by-products and impurities must be removed and this causes complexity in operation and increase of cost.
Therefore, in situ DNA synthesis reagents are needed, which do not require any steps of isolation and purification in preparation, and can be used as they are in a form of reacted solution for further reaction of synthesis of DNA oligomers.
Furthermore, the above conventional method has severe restrictions. One of them is that since accurate control of reaction on the solid phase is very difficult, particularly when the method is designed on a desired reaction scale, it is very difficult to set conditions therefor. Moreover, the porous glass is very expensive. Furthermore, since it is fundamental to use the reagents in excess amounts, this method is economically very disadvantageous when oligodeoxyribonucleotides are to be obtained in large amounts.
Specifically, it is technically not easy and requires much cost to design and practice the reaction in such a scale as exceeding 1 mmol utilizing a porous glass. These problems mean that if oligodeoxyribonucleotides were utilized for the pharmaceutical use, supply of them would be actually difficult.
In the production of oligodeoxyribonucleotides and oligoribonucleotides, there are demanded novel nucleoside compounds which can be utilized as starting materials for obtaining oligonucleotide in large amounts or as building blocks for extension of chains by use of easily available solid phase carriers, thereby facilitating separation and purification of intermediate products.
Furthermore, all of the steps in the above-mentioned conventional synthesis method are constructed of consecutive reactions and, hence, oligonucleotides having the desired sequences cannot be obtained unless all of the steps proceed with reaction yields of 100% or extremely close to 100%. Especially, the phosphorylation reaction step (condensation reaction) which is a step of extension of nucleotide chains gives a yield of 98.5-99.5 for each extension reation even in the present highest level, and this reaction yield determines the total yield of oligonucleotides having a desired sequence.
Recently, there is a report that a synthesis method in which a dimer nucleotide is used as a building block in building nucleotide chains thereby reducing the number of condensation is effective for improving the total yield (Krots et al, “Bioorg. Med. Chem. Lett.”, 1997, 7, 73-78). Furthermore, there are many chemical synthesis methods for oligonucleotides using dimers or higher as building blocks (for example, “Chemistry of Nucleic Acid and Molecular Biology-Elements of Chemistry 46” edited by Japan Chemical Association, and published by Gakkai Shuppan Center, 1985, pp.209-240). However, in the case of these building blocks, a series of the steps of protection-deprotection and phosphorylation are very complex and furthermore the operations such as extraction and chromatography are necessary in each step for the removal of by-products and impurities. These cause not only complexity of operation, but also increase the cost for the synthesis of building blocks.
Hitherto, the compound (V′) in the following formula (X) has been known as a nucleotide block which is an intermediate starting material for DNA oligomers, and as a method for synthesizing this compound, there is known a method which comprises the steps of reacting phosphoramidite compound (I′) with a nucleoside derivative (IX), oxidizing the nucleotide bond of the resulting nucleotide, and removing the protective group for 3′-position hydroxyl group from the resulting nucleotide derivative as shown in the following formula (X) (Refer to Japanese Patent Kohyo No.08-507752 corresponding to PCT/WO94/15946).
In the above formula (X), B
1
and B
3
are bases protected with protective groups common in nucleotide chemistry, A
1
and A
3
each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group, R
1
and R
4
each represents a protective group common in nucleotide chemistry, and X represents a dialkylamino group.
According to the above method, the nucleoside derivative represented by the formula (IX) which is a starting material must be produced in accordance with the reaction in the following formula (XI), and, in addition, the protective group R
4
must be removed from the nucleotide block derivative after dimerization reaction of nucleotide.
Therefore, in order to obtain a desired nucleotide block at high purity, the synthesis steps need many stages, and furthermore by-products or impurities must be removed thereby causing complexity of operation and increase of the cost.
In the above formula (XI), B
3
, R
1
, R
4
and A
3
are the same as defined in the formula (X).
Therefore, there are demanded a novel nucleotide compound and nucleotide block from which a nucleotide block useful for preparation of DNA oligomers can be simply produced without complex synthesis step and isolation and purification steps, as well as a method for producing an oligonucleotide using the same.
The object of the present invention is to provide nucleotide compounds and nucleotide blocks which meet the above-mentioned various demand, and a method for producing the same, and a method for producing an oligonucleotide using the nucleotide block.
As a result of intensive research conducted by the inventors in an attempt to solve the above problems, it has been found that when a nucleotide derivative represented by the following formula (I) is used, the above pr
Horie Yoji
Kitamura Akinori
Uchida Takayoshi
Yoshida Tadao
Toagosei Company, Ltd.
Wilson James O.
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