Nucleoside derivatives, and their use in oligonucleotide...

Details Nucleoside derivatives, and their use in oligonucleotide... Nucleoside derivatives, and their use in oligonucleotide...

1998-12-03

2001-02-13

Wong, Edna (Department: 1741)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025310, C536S025330, C536S025340, C536S025500, C536S028540, C536S028510, C536S027620, C536S027810, C536S026700, C536S026800, C536S027600, C544S243000, C544S244000, C205S427000

Reexamination Certificate

active

06187914

ABSTRACT:

The invention relates to reagents for oligonucleotide synthesis, and in particular to nucleoside derivatives and blocking reagents which can be used for the electrochemical synthesis of oligonucleotides.
Application PCT WO 94/22889 in the name of CIS BIO INTERNATIONAL (inventors TEOULE et al.) describes derivatives of electron conducting polymers (ECP) which can be used as a support for the synthesis of biological polymers, in particular of oligonucleotides. One of the uses of derivatives of electron conducting polymers which is described in this application is the addressed synthesis of oligonucleotides in situ on microelectrodes coated with an electron conducting polymer, which makes it possible, in particular, to obtain oligonucleotide matrices. The oligonucleotide or peptide matrices indeed constitute a very advantageous tool in the sequencing field, as well as in the diagnostic field (genetic diseases, infectious diseases, cancer, cell typing) or for the screening of active molecules.
During conventional methods of oligonucleotide synthesis on a solid support, nucleotides are used whose phosphate group, the exocyclic amine function of the A, C and G bases, and the 5′-OH group of the sugar bear protecting groups. To carry out each step for extending the oligonucleotide chain, the 5′-OH group of the sugar in the nucleoside from which the extension proceeds is deprotected so that the 5′-OH function liberated can react, in the presence of a coupling agent, with the phosphate group of the next nucleotide in order to form a phosphotriester bond (optionally via a phosphite which is then converted to a phosphotriester by oxidation). The cycle is then repeated: deprotection of the 5′-OH group/coupling of the next nucleotide via a phosphotriester bond, until the oligonucleotide of the desired size is obtained. At the end of each cycle, the unreacted 5′-OH functions are blocked by esterification; this step is called <<capping>>.
The protection of the A, C and G bases is designed to avoid spurious reactions involving the exocyclic amine functions of these bases. The protecting groups for these bases, as well as those for the phosphate group, are cleaved at the end of the oligonucleotide synthesis.
The use of an electron conducting polymer as a synthesis support makes it possible to carry out the synthesis of oligonucleotides using one or more steps for electrochemical cleavage of the different protecting groups. This electrochemical deprotection is achieved by creating a potential difference between the working electrode and a counter-electrode.
The use of microelectrodes arranged in the form of a matrix as a solid support for the in situ synthesis of oligonucleotides makes it possible to carry out, for the addition of a given nucleotide, the selective electrochemical deprotection of the 5′-OH function of the developing oligonucleotide chains on some of the electrodes of the matrix; this therefore makes it possible to add the relevant nucleotide only to the desired oligonucleotide chains and to obtain, at the end of the synthesis, oligonucleotide matrices bearing, at different points, oligonucleotides of different sequences.
Application PCT WO 94/22889 cited above, thus describes the use of a thiopixyl group or of a p-nitrobenzoyl group as electrolabile protecting group for the 5′-OH function of the sugar.
In order to be in a position to synthesize oligonucleotides with a variety of sequences using one or more electrochemical deprotection steps, it is advisable to use, for all the functions to be protected, mutually compatible protecting groups.
Accordingly, it should be possible for the electrolabile protecting group for the 5′-OH function of the sugar to be cleaved at a voltage which does not affect either the stability of the nucleosides or that of the protecting groups for the exocyclic amine function of the A, C and G bases. As regards the latter, they should not modify the electronic properties of the base, which would risk influencing its reactivity or the stability of the N-glycoside bond. Protecting groups should therefore be selected which do not destabilize the nucleosides in relation to the reagents for oligonucleotide synthesis, or in relation to reactive species generated during the application of the deprotection voltage, and which, in addition, allow sufficient solubility of the nucleoside.
Likewise, the protecting group for the phosphorus in 3′ should be stable under the conditions used for the electrochemical deprotection of the sugar and of the bases.
Finally, the ester formed during the capping in order to block the unreacted 5′-OH functions should Ad 11also be stable under these conditions.
The inventors have now developed nucleoside derivatives bearing protecting groups which satisfy all the abovementioned conditions, and in particular which make it possible to easily carry out an electrochemical deprotection during the synthesis of oligonucleotides. The subject of the present invention is the use, in a method for the synthesis of oligonucleotides comprising at least one electrochemical step, of at least one nucleoside derivative corresponding to the formula (I) below:
in which
R
1
represents:
an electrolabile group corresponding to the following formula (IIa):
in which R
4
represents an NO
2
group and R
5
represents a hydrogen atom, a linear or branched linear or branched C
1
to C
10
alkyl group, a group
or a group
where R′ represents a linear or branched C
1
to C
10
alkyl group, or a group —O—R′ where R′ represents a C
1
to C
10
alkyl group, or alternatively
an electrolabile group corresponding to the following formula (IIb):
in which one of the groups Ra, Rb or Rc represents a phenyl group, or a substituted phenyl group, and the other two represent hydrogen atoms,
R
2
represents a hydrogen atom, or a phosphoramidite, phosphonate or phosphotriester group;
R
3
represents a hydrogen atom, a hydroxyl radical, a substituted hydroxyl radical or a halogen.
B represents a radical derived from a purine or a pyrimidine base corresponding to one of the following formulae:
where R
6
represents a protecting group for the amine function, other than as isobutyryl or phenylpropionyl group, corresponding to one of the formulae (VII), (VIII) and (IX) below:
in which R
9
and R
11
, which may be identical or different, represent a hydrogen atom, or a C
1
-C
10
alkoxy or alkyl group, R
12
represents a linear or branched C
1
-C
10
alkyl radical, and R
10
is chosen from the radicals of formula:
R
7
represents a protecting group for the amine function, other than a phenylpropionyl group, corresponding to one of the formulae (VII), (VIII) and (IX) defined above;
R
8
represents a protecting group for the amine function, other than an isobutyryl, phenylpropionyl or phenoxyacetyl group, corresponding to one of the formulae (VII), (VIII) and (IX) defined above;
with the exception of the derivatives in which R
1
is an electrolabile group of formula (IIa) in which R
4
represents an NO
2
group and R
5
a hydrogen atom, and B represents thymine.
Preferred derivatives are those in which R
1
corresponds to the formula (IIa), in which R
4
represents an NO
2
group and R
5
represents a hydrogen atom, and among these, derivatives in which:
B corresponds to the formula IV in which R
6
is an anisoyl group, or alternatively
B corresponds to the formula V in which R
7
is a benzoyl group or a phenoxyacetyl group, or alternatively
B corresponds to the formula VI in which R
8
is a t-butylphenoxyacetyl group.
The abovementioned nucleoside derivatives can undergo, without alteration, all the oligonucleotide synthesis operations, and in particular the application of the electric voltage necessary for cleaving the electrolabile protecting groups.
They are therefore particularly appropriate for use for the synthesis of oligonucleotides on supports coated with electron conducting polymers, in particular microelectrodes, or microelectrode matrices.
It is also possible, in the same man

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