Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1998-06-19
2001-03-27
Marschel, Ardin H. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C536S025300
Reexamination Certificate
active
06207797
ABSTRACT:
This application was filed under 35 U.S.C. §371 from PCT/FR96/02003, filed Dec. 13, 1996.
The present Invention relates to the treatment of polymers synthesized electrochemically, for the purpose of using them for the construction of addressed matrices.
Matrices of compounds having an affinity for biological products, for example matrices of peptides, of oligonucleotides, of receptors, and the like, can be constructed in several ways. Three main methods have been described: photochemically directed deposition [Fodor S. et al., Science, 251, 767-773 (1991)], mechanical deposition [Khrapko K. R. et al., DNA Sequencing and Mapping, 1, 375-338 (1991)] or electrochemically directed deposition [PCT International Application published under the number WO 94/22889; Livache et al., Nucl. Acids. Res., 22, 15, 2615-2921 (1994)].
Oligonucleotides can be deposited on different points of the support, either by in situ nucleotide synthesis [Fodor S. et al.; PCT Application WO 94/22889] or by successive attachments of presynthesized oligonucleotides [Fodor S. et al.; Khrapko K. R. et al.; PCT Application WO 94/22889].
In the first case, the entire surface of the support is washed after each addition of nucleotide. This washing operation serves simply to remove the excess reagent present on the support; it is in no way linked to the actual nature of the support.
In the second case (successive attachments of oligonucleotides), two types of treatment are described; some writers [Saiki R. K. et al., Proc. Natl. Acad. Sci. USA, 89, 6230-6234 (1989), Khrapko et al. (already cited) or Lamture et al., Nucl. Acids. Res., 22, 2121-2125 (1994)] carry out a single final washing of the support after the attachment of all the oligonucleotides. This washing with water or in a hybridization buffer makes it possible to remove the probes which have not reacted on the support. With the same aim, Livache et al. rinse the support with water between each attachment of oligonucleotide; in this case, there are therefore as many rinsing operations as there are coupling operations of oligonucleotides.
However, the Inventors have found that, when several depositions of polymers produced electrochemically are carried out at different points of the same electrode matrix, sporadic interelectrode contaminations can take place. Indeed, during the synthesis of a polymer on the electrode number n in the presence of the n oligonucleotide, contamination by the n oligonucleotide on the n−1 electrode, already covered by an n−1 oligonucleotide, can be observed. This results in a weakly positive signal of the n−1 spot during hybridization with the n oligonucleotide. This phenomenon can be illustrated by the results of four cross-hybridizations cited in the publication by Livache et al., Nucl. Acids. Res., 22, 15, 2615-2921 (1994) . Two slight contaminations (approximately 10% of the maximum signal) are visible on the four deposits. They had been interpreted at the time as being non-specific hybridizations; however, subsequent experiments by the Inventors, involving different oligonucleotide sequences, have shown that they were in fact contaminations.
It is important, for diagnostic use of a matrix of oligonucleotides which are coupled to a support, for this type of contamination to be completely removed. Now, the washing treatments generally employed are insufficient to produce this result.
The present Invention had as aim the specific treatment of polymers produced electrochemically, in order to decrease, indeed eliminate, the sporadic phenomena of contamination.
With this aim, the Inventors studied the mechanism of electrochemical polymerization. It is known that this polymerization in particular involves radical reactions [Genies et al., J. Electroanal. Chem., 149, 101-113 (1993)] and, when an n−1 polymer is synthesized, it may possibly retain at the surface, for a certain period of time, reactive species capable of capturing products during the polymerization on the n electrode.
The Inventors have put forward the hypothesis that, by reducing the surface reactivity of the polymer, success would be achieved in removing the contaminations observed during the addressed synthesis of biological molecules at the surface of the said polymer.
The subject of the present Invention is a method for the synthesis of a copolymer by electrochemical copolymerization, characterized in that it comprises a treatment which makes it possible to reduce the surface reactivity of the said copolymer.
In particular, the subject of the present Invention is a method for preparing a copolymer of following general formula (I):
by electrochemical copolymerization of a compound of following general formula (II):
with a compound [A]
[A] representing a monomer unit of a polymer capable of being produced by electrochemical copolymerization, [B] representing a side group, which can be a monomer or a polymer, bonded to the unit [A] by a covalent bond or via a spacer arm, and x and y representing integers equal to or greater than 1, which method is characterized in that it comprises, on conclusion of the said electrochemical copolymerization, at least one stage where the copolymer produced is subjected to a treatment which makes it possible to reduce its surface reactivity.
Within the meaning of the present Invention, “surface reactivity” is understood to mean the persistence, at the surface of the copolymer formed, of reactive species capable of attaching a unit of formula (II) and “reduction of the surface reactivity” is understood to mean the at least partial inactivation of these reactive species which makes possible the attachment of only a zero or low amount of units of formula (II). The contaminations resulting from this attachment are thus eliminated or reduced until they are no longer detectable or until they only produce a negligible signal, readily distinguishable from a positive signal.
According to a preferred embodiment of the present Invention, [A] represents a monomer of electronically conductive polymer; the said electronically conductive polymer is preferably chosen from polyacetylene, polyazine, poly(p-phenylene), poly(p-phenylene vinylene), polypyrene, polypyrrole, polythiophene, polyfuran, polyselenophene, polypyridazine, polycarbazole, polyaniline and their substituted derivatives (the substituents can be, for example, saturated or unsaturated hydrocarbon chains (preferably C
1-3
chains), aromatic or non-aromatic rings, or heterocycles, and the like); the said electronically conductive polymer can also be a copolymer of two or more different monomers [A], for example pyrrole monomers and thiophene monomers.
Advantageously, [A] is a pyrrole unit.
According to a preferred embodiment of the present Invention, [B] represents a nucleotide, an oligonucleotide or one of their derivatives or analogues.
In the context of the description of the present Invention, nucleotide derivative or analogue, for example, is understood to mean modified nucleotides, such as those described by Uhlmann [Chemical Review, 90:4, 543-584 (1990)]; nucleotide analogues forming part of the composition of synthetic oligonucleotides; nucleotide derivatives carrying protected functional groups which are commonly used in the synthesis of nucleic acids.
[B] can also be a non-natural compound which can be hybridized with nucleic acids, such as those described by Uhlmann (abovementioned publication).
The Inventors have found that three types of treatment could be employed to reduce the reactivity of the polymers:
a chemical treatment, based on products known for their antiradical activity or their inhibitory activity on radical polymerization;
an electrochemical treatment which makes it possible to reduce or to overoxidize the polymer;
rinsing in the presence of products capable of removing the short growing polymer chains adsorbed on the support.
According to a preferred embodiment of th
Livache Thierry
Roget Andre
Teoule Robert
Cis Bio International
Marschel Ardin H.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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