Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-22
2002-01-01
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S022100, C536S025310
Reexamination Certificate
active
06335438
ABSTRACT:
TECHNICAL FIELD
This invention concerns support matrices comprising a polyvinyl backbone [(—CH
2
CH
2
)
n
] to which amino groups and/or derivatized amino groups are attached via link structures.
The expressions “a derivatized amino group” and “a group derived from an amino group” means that the amino group has been chemically transformed to a group in which the nitrogen remains bound to a link structure.
In earlier known support matrices cross-linking structures and groups containing pure amino groups and derivatized amino groups have replaced hydrogens in the polyvinyl backbone. Hydrogens have also been replaced by other groups, such as alkyls (methyl, ethyl etc), various forms of aryls (such as phenyl, vinyl phenyl, ethyl phenyl etc), acyloxy, aryloxy, alkoxy etc. The groups have been further derivatized.
This type of support matrices can be used as a solid phase in adsorption and partition processes, such as chromatography, and in solid phase organic synthesis, and as support in cell culturing and for catalysts, such as enzymes.
This type of support matrices has previously been prepared by copolymerising mono-vinyl compounds with di-, tri and polyvinyl compounds, with at least one of the monomers containing a functionality that can be transformed to an amino group after the polymerisation. One alternative has been to copolymerise chloromethyl styrene with divinyl benzene followed by treatment with ammonia. Another alternative has been conversion of residual vinyl groups to groups containing an amine function.
It has also been suggested that similar support matrices can be produced by ammonia gas plasma treatment of various polymers containing a polyvinyl backbone. See for instance U.S. Pat. No. 5,369,012. Gas phase plasma treatment gives a more or less random introduction of functional groups on carbon atoms in the backbone or in pending groups.
Another possible method of making similar support matrices is nitration of cross-linked polystyrenes followed by reduction of the nitro groups to amino groups. Nitrations are generally carried out under very harsh conditions leading to a number of side reactions like oxidations. The subsequent reduction step is characterized by relatively low yields resulting in unreacted nitro groups on the polystyrene. In highly cross-linked materials these reactions are very difficult to use successfully. Unwanted nitro groups attached directly on the polyvinyl backbone are obtained in side reactions.
Previously known support matrices thus have been relatively cumbersome to manufacture because of the extra steps necessary for introducing the amine functions and of the side reactions occurring.
For porous matrices carrying amino groups, we have found that the availability of the amino groups in a given matrix often vary. This has not been considered optimal for certain applications, for instance solid phase synthesis of oligonucleotides and adsorption processes.
Commercially available support matrices comprising polyvinyl backbones for use in solid phase synthesis of oligonucleotides are sold with the first monomer attached to the matrix. The loading has been about 90 &mgr;mole of the first nucleotide per gram matrix (Primer Support High Load 30; Amersham Pharmacia Biotech AB, Uppsala, Sweden).
If not otherwise specified, the terms nucleotide, dexoxynucleotide, amino acid residue, DNA, RNA, oligo-/polypeptide includes their respective synthetic analogue, such as PNA (WO 9220703) and LNA for DNA.
The terms nucleotide, dexoxynucleotide, amino acid residue, DNA, RNA, oligo-/polypeptide, synthetic analogues thereof such as PNA (WO 9220703), LNA etc includes, if not otherwise specified.
OBJECTS OF THE INVENTION
A first object is to provide a simplified method for manufacturing support matrices carrying amino groups and/or derivatized amino groups, both of which are bound via a link structure to a polyvinyl backbone.
A second object is to provide support matrices that have an improved availability of amino groups and of derivatized forms thereof.
A third object is to provide improved support matrices for solid phase synthesis of oligonucleotides and oligopeptides, said matrices being improved in accordance with the first and second objects and leading to improved yields of a desired oligonucleotide/oligopeptide.
A fourth objective is to provide improved support matrices of the above-mentioned type for use in solid phase synthesis of organic compounds, the improvement being a loading (covalent attachment) of ≧100 &mgr;mole, such as ≧150 &mgr;mole or even ≧200 &mgr;mole of an organic reagent per gram dry matrix. These figures in particular refer to support matrices in which the organic reagent is a nucleotide, deoxynucleotide, or an analogue thereof.
A fifth object is to provide improved support matrices for the solid phase synthesis of organic compounds, with particular emphasis of combinatorial libraries of small organic molecules
THE INVENTION
It has now been recognized that these objects can be met in case one or more amino-(C
0-10
)hydrocarbon vinyl aromatic monomers, optionally having the amino group acylated, are copolymerised with other vinyl monomers. In other words essentially all amino groups in the matrix obtained after polymerisation will be of the same type, i.e. either a pure amino group or an acylated form thereof. Essentially no nitro groups or groups derived from side reactions occurring during nitration or during a subsequent reduction of the formed nitro groups to amino groups will be present in the matrix if the corresponding phenyl substituted polyvinyl polymer is nitrated.
The first aspect of the invention thus encompasses a method for the manufacture of a support matrix to which amino groups and/or acylated forms thereof are attached. The method comprises the step of copolymerising one or more monovinyl monomer (monomer I) with one or more di- tri- or polyvinyl monomers (monomer II). The method is characterized in that a part of the vinyl monomers carries an amino-(C
0-10
)hydrocarbon group or corresponding amido group (acylated amino) attached to an aromatic ring (monomer III). After the polymerisation the polymer material formed may be collected and further processed. The amino groups and/or acylated forms thereof may subsequently be transformed to a desired functionality. The amino groups may be primary, secondary or tertiary.
The amino-(C
0-10
)hydrocarbon group is preferably a pure amino group, such as in amino monovinyl benzenes (for instance amino styrene) and amino vinyl naphthalenes, i.e. with the hydrocarbon group being non-existent. The amino group may be derivatized, such as in amides.
The hydrocarbon group, if present in the amino-(C
0-10
) hydrocarbon group may be straight, branched or cyclic. It may be aromatic or non-aromatic, such as containing a phenylene or consisting of a pure alkylene chain, respectively.
Monomer III is part of monomer I and/or monomer II.
Monomer III may be isoforms of amino-(C
0-10
)hydrocarbon vinyl aromatics in which a vinyl group and an amino-(C
0-10
) hydrocarbon group are orto, meta or para to each other. Monomer III may also comprise amino-(C
0-10
)hydrocarbon vinyl naphthalenes in which either of the vinyl group or the amino-(C
0-10
) hydrocarbon group is in the 1- or 2-position.
The acylated amino group in monomer III complies with the formula
—NR
1
R
2
in which R
1
and R
2
are selected among
a) hydrogen,
b) straight, branched and cyclic hydrocarbon groups or corresponding acylated forms.
Each of the hydrocarbon groups may have a hydrocarbon chain of 1-20 carbon atoms, optionally substituted with one or more hydroxy, alkoxy or amino groups and containing a nitrogen, an oxygen or a sulphur atom replacing a carbon atom in the chain.
More specifically R
1
and R
2
encompass alkyls, aralkyls arylalkyls and aryls, for instance substituted or unsubstituted phenyl containing none, one or more heteroatoms, such as nitrogen or oxygen. Alkoxy is preferably C
1-7
alkoxy. The nitrogen of an amino group that may be present in an R
1
or R
2
may carry one or two hydrocarbo
Birch & Stewart Kolasch & Birch, LLP
Riley Jezia
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