Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2003-01-28
2004-11-30
Peselev, Elli (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S025340, C536S023100, C536S023200, C536S025300, C432S103000, C110S345000, C435S193000, C424S452000, C424S464000, C424S465000, C424S489000, C252S182160, C422S131000, 56
Reexamination Certificate
active
06825339
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for preparing polynucleotides on a solid support. The present invention also relates to a reactor containing a solid support and to a device including this reactor, which are useful in the process for preparing polynucleotides according to the invention.
BACKGROUND OF THE INVENTION
The synthesis of polynucleotides on a solid support is particularly used in automated syntheses of DNA or RNA oligonucleotides. In the present application, “polynucleotides” is understood to mean deoxyribonucleic acid or ribonucleic acid fragments or, more generally, polynucleotides or oligonucleotides where the bases, internucleotide phosphate linkages, or alternatively the ribose rings of the bases, can be chemically modified in a known manner. This may be especially oligonucleotides with &agr; or &bgr; anomers, oligonucleotides with inter-nucleotide linkage of the phosphorothioate or methyl phosphonate type, or alternatively oligothionucleotides.
The principle for the chemical synthesis of nucleic acids on a solid support is nowadays widely described in the specialist literature, and a number of apparatus are available on the market which perform automatically all or part of the synthesis steps. Among the chemical routes described, only the so-called phosphoramidites method (Caruthers et al.: EP 0,035,719 BI) is up until now sufficiently efficient to envisage the production of nucleic acids on an industrial scale.
The preparation of oligonucleotides or polynucleotides is carried out in a reactor containing a solid support and comprises the treatment of the solid support such as an inorganic polymeric support by a series of successive steps, each of the series leading to the addition of a new nucleotide on the support. The series of successive steps, or synthesis cycles, are carried out as many times as is required by the manufacture of an oligonucleotide or a polynucleotide of desired length.
In a process for synthesizing polynucleotides on a solid support, the solid support traditionally consists of glass beads of controlled porosity (CPG) or, more generally, of particles of a functionalized inorganic or organic polymer.
The techniques conventionally used involve the use of eight different reagents as solid supports, consisting of the said functionalized inorganic or organic polymer linked to a nucleoside A, T, C, G or U, depending on whether the sequence to be prepared contains, as first deoxyribo- or ribonucleotide A, T, C, G or U. Manufacturers therefore supply reactors in which one of these nucleosides has previously been attached to the support. Depending on whether the sequence starts with A, T, C, G or U, the appropriate reactor is then chosen. The elongation of this first nucleoside then occurs in the 3′→5′, or 5′→3′ direction, using coupling reagents.
Numerous supports have already been described in the literature for the solid phase synthesis of oligonucleotides.
There may be mentioned organic polymers such as polystyrene (Nucleic Ac.1980, volume 8), polyacrylamide acryloylmorpholide, polydimethylacrylamide polymerized on kieselguhr (Nucleic Ac. 9(7):691 (1980)).
Other supports described are of inorganic nature, in particular based on silica functionalized by a hydrocarbon radical carrying an NH
2
and/or COOH group (J. Am. Chem., 105:61 (1983)), or the support based on silica functionalized by a 3-aminopropyltriethoxysilane group whose use in phosphite and phosporamidite synthesis for the preparation of oligonucleotides was described for the first time in European Patent No. 0,035,719.
There is known, from French Patent Application FR 93 08 498 and PCT/FR94/00842, a process for the solid phase synthesis of oligonucleotides in which a so-called “universal” support is used, that is to say a solid support which can be used regardless of the first nucleotide of the RNA or DNA to be synthesized, regardless of the type of monomeric reagent used during the synthesis, that is to say regardless of the type of substitution on the phosphate group in 3′ or in 5′, depending on whether the synthesis is carried out in the 5′→3′ or 3′→5′ direction.
In particular, the “universal nature” of the solid phase supports can be obtained by functionalization of the inorganic or organic polymer with a hydrocarbon radical containing glycol type groups in which an OH group and a nucleophilic group are present in the vicinal position, that is to say on two adjacent carbons, at the end of the hydrocarbon radical, it being possible for these two carbons to be optionally substituted by inert groups. “Inert group” is understood here to mean a group which does not react under the conditions encountered during the various steps of the synthesis on a solid support of nucleic acids according to the invention.
In a specific embodiment, a process for synthesizing polynucleotides comprises the following steps of:
1) condensing the OH group in 5′ or 3′ of the first nucleotide or of an oligonucleotide linked at its other 3′ or 5′ end to the said solid support, by means of a coupling agent, with the phosphate group optionally substituted respectively in position 3′ or 5′ of a monomeric nucleotide reagent protected in 3′ and 5′;
2) oxidizing or sulfurizing the phosphite type internucleotide linkage obtained in step 1) into a phosphate linkage respectively;
3) deprotecting the 5′-O or 3′-O end of the product obtained in step 2);
4) repeating steps 1) to 3) as many times as there are nucleotides to be added in order to synthesize the nucleic acid.
The above steps lead to an oligonucleotides linked to the solid support. The process comprises a final step of detaching the nucleic acid from the support and removing the groups for protecting the bases and, where appropriate, the 2′-O positions of the nucleic acid.
In the techniques where the solid support is already linked to a first nucleoside corresponding to the first nucleotide of the sequence to be prepared, before the start of the synthesis cycles, the said support generally contains a protection in 5′ or 3′ of the said nucleoside. In this case, the synthesis cycle starts with a deprotection step in acidic medium, in general a detritylation.
According to the variants used most commonly, the said monomeric nucleotide reagent corresponds to the formula:
in which:
A represents H or an optionally protected hydroxyl group,
is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected,
C is a conventional protective group for the 5′-OH functional group,
x=0 or 1 with a) when x=1:
R
3
represents H and R
4
represents a negatively charged oxygen atom, or
R
3
is an oxygen atom and R
4
represents either an oxygen atom or an oxygen atom carrying a protecting group, and
b) when x=0, R
3
is an oxygen atom carrying a protecting group and R
4
is either a hydrogen or a disubstituted amine group,
when x is equal to 1, R
3
is an oxygen atom and R
4
is an oxygen atom, the method is in this case the so-called phosphodiester method; when R
4
is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method,
when x is equal to 1, R
3
is a hydrogen atom and 4 is a hydrogen atom and R
4
is a negatively charged oxygen atom, the method is in this case the so-called H-phosphonate method, and
when x is equal to 0, R
3
is an oxygen atom carrying a protecting group and R
4
is either a halogen, the method is in this case the so-called phosphite method and, when R
4
is a leaving group of the disubstituted amine type, the method is in this case the so-called phosphoramidite method.
The steps of a cycle of synthesis by the phosphoramidite method are conventionally the following:
1) condensation of the 5′ terminal hydroxyl of a nucleoside or of an oligonucleotide covalently attached to the solid support with a phosphite type compound according to the reaction:
2) oxida
Chatelain François
Kumarev Viktor
Khare Devesh
Peselev Elli
Proligo LLC
Swanson & Bratschun L.L.C.
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