Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – From phenol – phenol ether – or inorganic phenolate
Reexamination Certificate
2002-03-26
2004-06-15
Hightower, P. Hampton (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
From phenol, phenol ether, or inorganic phenolate
C528S310000, C528S323000, C528S328000, C528S332000, C528S342000, C528S367000, C528S368000, C528S369000, C528S480000, C528S486000, C530S333000, C530S334000, C530S335000, C530S337000, C530S338000, C530S341000, C562S442000, C562S450000, C525S054100, C525S054110
Reexamination Certificate
active
06750312
ABSTRACT:
The present invention relates to a method of preparing support materials useful in solid phase chemical synthesis processes together with the solid materials produced thereby and intermediates used in the process. The support materials are useful in solid phase synthetic processes for the production of a variety of organic molecules, in particular peptides.
The multi-stage synthesis of an organic molecule typically involves numerous isolation steps to separate intermediates, produced at each stage, before progressing to the subsequent stage. These intermediates often require purification to remove excess reagents and reaction by-products and will include procedures such as precipitation, filtration, bi-phase solvent extraction, solid phase extraction, crystallisation and chromatography.
If the reaction chemistry is well defined, many of the isolation procedures used in solution phase synthesis are avoided by reversibly attaching the target molecule to a solid support in a way analogous to the use of a protecting group in a traditional synthesis. Excess reagents and some of the side-products can thereby be removed by filtration and washing of the solid support. Providing that the reactions are efficient and no solid support is lost, the target molecule is recovered in essentially quantitative yield, an objective rarely achieved in solution phase synthesis. In addition the time required to perform operations on a solid support is generally accepted to be a fifth of that required to carry out the equivalent stage in a solution phase synthesis. Another advantage of the solid phase approach is that the whole assembly is carried out in a single reactor.
There are disadvantages to the solid phase approach however. In particular, commercially available supports commonly used for solid phase synthesis of peptides allow a comparatively low loading (<1mmol/g) of reagent, resulting in reactions being carried out at a higher dilution than would normally be achievable in solution. To counteract this, reagents used to carry out the stepwise solid phase assembly are normally used in large excess (3-6 equiv.) and although the excesses are readily removed in the solid phase approach this can add an unnecessary burden on the economics of the process.
Some improvements in terms of increasing the loading of the solid support and solid phase peptide synthesis have been reported. Epton, R. et al; (1985);
Int. J. Biol. Macromol
., 7, 287-298 describes the use of a bead-form phenolic core polymer as a support matrix. Modified forms of this matrix material, in which the phenolic core is condensed with protected tyrosine are described by Epton, R. et al., in
Peptides
1986
, Proceedings of the
19
th
European Peptide Symposium
, Ed., Theodoropolulos, D.; Publ., Walter de Gruyter, Berlin, 1987, p151-154.
Kates et al., Peptide Science, 47, 5, 1998, 365-380 describes the introduction of polyethylene glycol into solid phase supports to increase the hydrophilicity of the support. Ornithine which is differently protected on each amino group was added to amino functionalised polystyrene, and one protecting group removed so as to allow for bond formation with polyethylene glycol (PEG) by way of a carboxylic acid group which is thereby introduced into the chain. This increases the number of available linking groups, but the loading of the resins was only 0.3-0.5 mmol/g. Solid phase dendrimer synthesis involving repeated treatments of a functionalised support with methyl acrylate and 1,3-propanediamine to produce a structure branched as a result of the production of tertiary amine groups has been described by Wells et al., Peptide Science, 47 (1998) 385-396.
The applicants have found a method by which a support can be modified using simple process steps to significantly increase the loading capacity, in particular for peptide synthesis, where it is particularly suitable and economic for large scale manufacture of peptides.
The present invention provides a method for preparing a solid support material for carrying out a chemical reaction, the said method comprising the following steps:
(i) reacting an amino functionalised solid material with a carboxylic acid having at least two similarly protected amino groups to form amide bonds between them,
(ii) removing the protecting groups in a single step,
(iii) optionally repeating steps (i) and (ii) one or more times, using the product of the preceding step as the amino functionalised solid material, and
(iv) connecting a linkage agent to at least some of the free NH
2
groups of the product.
The product of this method is an amino functionalised branched amide-containing organic structure with the amino groups connected to a linkage agent.
This method provides a simple means of increasing the loading capacity of a support material without the need for complex chemical processing. The number of sites available to attach to linkage agents increases as a result of the production of branches. The number of branches available in step (iv) will depend upon the number of NH
2
groups present in the final product, and this is a function of the number of free NH
2
groups within the carboxylic acid structure and the number of times step (iii) is repeated.
The carboxylic acid used in the process is suitably an amino acid containing more than one amino group. Conveniently, this may comprise a naturally occurring amino acid such as lysine or ornithine but synthetic acids may also be used.
Suitable protecting groups for use in the process would be understood in the art, as would the means by which they may be added and subsequently removed. Examples of suitable protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc). Protected forms of amino acids such as Fmoc and Boc protected forms are known in the art or they can be prepared using conventional methods.
The coupling reaction (i) and the reaction of step (iv) above are suitably carried out in an organic solvent such as N,N-dimethylformamide (DMF) or N-methylpyrrolidinone (NMP) in the presence of a coupling reagent. Coupling reagents include those known in the art of peptide synthesis, see for example those coupling reagents disclosed by Wellings, D. A.; Atherton, E.; in
Methods in Enzymology
, Publ., Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N,N′-diisopropylcarbodiimide (DIC), and reagents that form active esters, particularly benzotriazole active esters in situ, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or benzotriazole-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), optionally in the presence of a base such as diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM); or any other suitable activating agent common in the art of peptide synthesis.
The deprotection reaction conditions used in step (ii) will depend largely on the nature of the particular protecting group used and will be readily apparent to chemists. For instance, reaction with a base such as piperidine will result in the removal of protecting groups such as Fmoc groups and the reaction is suitably effected in an organic solvent such as N,N-dimethylformamide (DMF) or N-methylpyrrolidinone (NMP).
As used herein, the term “branched amide-containing organic structure” describes organic moieties which have a plurality of optionally substituted hydrocarbyl chains, each of which may be for example of from 2 to 12 suitably from 2 to 8 carbon atoms in length, and may be optionally interposed by a heteroatom, such as oxygen, nitrogen and sulphur. At least some of the chains are linked together by way of amide bonds, formed during the method of the reaction. Each chain of hydrocarbyl atoms may itself be branched. At least some, and preferably substantially all of the chains of the branched structure will carry a linkage agent or a protected form thereof.
Optional substituents on the hydrocarbyl chains may include any group which does not interfere with the subsequen
Brown Richard John
Harris Craig Stephen
Montgomery Francis Joseph
Wellings Donald Alfred
Avecia Limited
Hampton Hightower P.
Pillsbury Winthorp LLP
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