Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1998-12-28
2001-08-07
Naff, David M. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C435S068100, C435S181000, C530S402000, C530S816000
Reexamination Certificate
active
06271345
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to enzymatically cleavable linkers for solid-phase syntheses, to a process for their preparation and to their use.
2. Description of the Related Art
A large number of molecular assay systems are being developed for modern research looking for active substances, such as receptor binding assays, enzyme assays and cell-cell interaction assays. Automation and miniaturization of these assay systems makes it possible to assay an increasingly large number of chemicals for their biological effect in random screening and thus form a possible use as lead structure for an active substance in medicine, veterinary medicine or crop protection.
This development has led to classical synthetic chemistry becoming the limiting factor in research looking for active substances.
If the efficiency of the developed assay systems is to be fully exploited there must be a considerable increase in the efficiency of chemical synthesis of active substances.
Combinatorial chemistry can contribute to this required increase in efficiency, especially when it makes use of automated solid-phase synthetic methods (see, for example, review articles in J. Med. Chem. 37 (1994), 1233 and 1385).
The principle of these combinatorial syntheses is based on reaction at every stage of the synthesis not just with one building block in the synthesis but with several, either in parallel or in a mixture. All possible combinations are formed at every stage, so that a large number of products, called a substance library, results after only a few stages with relatively few building blocks.
Solid-phase synthesis has the advantage that byproducts and excess reactants can easily be removed, so that elaborate purification of the products is unnecessary. Reaction rates can be increased, and conversions optimized, by large excesses of the dissolved reactant. The finished synthetic products can be passed directly, i.e. bound to the support, or after elimination from the solid phase to mass screening. Intermediates can also be tested in the mass screening.
Applications described hitherto are confined mainly to the peptide and nucleotide sectors (Lebl et al., Int. J. Pept. Prot. Res. 41, 1993: 203 and WO 92/00091) or their derivatives (WO 96/00391). Since peptides and nucleotides have only limited uses as drugs because of their unfavorable pharmacological properties, it is desirable to utilize the solid-phase synthetic methods known and proven in peptide and nucleotide chemistry for synthetic organic chemistry.
U.S. Pat. No. 5,288,514 reports one of the first combinatorial solid-phase syntheses in organic chemistry outside peptide and nucleotide chemistry. U.S. Pat. No. 5,288,514 describes the sequential solid-phase synthesis of 1,4-benzodiazepines.
WO 95/16712, WO 95/30642 and WO 96/00148 describe other solid-phase syntheses of potential active substances in combinatorial chemistry.
However, in order fully to utilize the possibilities of modern assay systems in mass screening, it is necessary continually to feed novel compounds with a maximum degree of structural diversity into the mass screening. This procedure makes it possible to reduce considerably the time taken to identify and optimize a novel lead structure for active substances.
It is therefore necessary continually to develop novel and diverse combinatorial solid-phase syntheses.
It is important for these novel syntheses that the individual building blocks in the solid-phase synthesis are optimally matched with one another. The choice of the solid phase, such as glass, ceramic or resins, and of the linker crucially influences the subsequent chemistry on the support.
In order to be able to carry out the widest possible range of organic syntheses on solid phases there is a considerable need for novel solid phases, and novel linker and anchor groups, to be developed.
Linker groups used hitherto are labile to bases or acids, and their elimination conditions are too drastic for many substances synthesized on the support. Great efforts are therefore being made to construct linkers which can be eliminated from the solid phase under milder conditions.
It would be desirable in this connection to be able to use enzymes for cleavage of the linkers under mild conditions, as is already possible in a few cases for protective groups. An example of an enzymatically cleavable protective group is described by Waldmann et al. in Angew. Chem. 107 (1995) 2425-2428.
Elmore et al. describe a first enzymatically cleavable linker for solid-phase peptide synthesis (J. Chem. Soc., Chem. Commun. 14 (1992) 1033-1034) which can be cleaved off the support under mild conditions. Schuster et al. describe another enzymatically cleavable linker for solid-phase syntheses of sugars (J. Am. Chem. Soc. 116 (1994) 1135-1136 and U.S. Pat. No. 5,369,017).
The disadvantage of both methods is that parts of the linker always remain in the product after the enzymatic cleavage. In addition, both methods are greatly restricted with regard to the linker-cleaving enzymes; thus Elmore uses calf spleen phosphodiesterase for the cleavage, and Schuster et al. describe serine proteases for the cleavage.
SUMMARY OF THE INVENTION
It is an object of the present invention to develop a linker which can be cleaved under mild conditions and, does not have the abovementioned disadvantages and makes possible a wide range of solid-phase organic syntheses.
We have found that this object is achieved by an enzymatically cleavable linker which is bound to a solid phase and on which organic compounds are synthesized via a functional group, wherein the linker contains a recognition site for a hydrolytic enzyme and is fragmented by reaction with the enzyme in such a way that no parts of the linker molecule remain in the synthesized product, and wherein the recognition site for the enzyme and the site at which the synthetic product is liberated by fragmentation of the linker are different.
The invention additionally relates to the preparation of the linkers and to their use.
DETAILED DESCRIPTION OF THE INVENTION
A preferred linker has the formula I
in which the variables and substituents have the following meanings:
(P) a solid phase
(S) a spacer with a length equivalent to 1 to 30 methylene groups
R hydrogen or a radical which is inert under the reaction conditions or two adjacent inert radicals R which together form an aromatic, heteroaromatic or aliphatic ring
R
1
substituted or unsubstituted C
1
-C
20
-alkyl, C
3
-C
20
-alkenyl, C
3
-C
6
-alkynyl, C
l
-C
20
-alkylcarbonyl, C
1
-C
20
-alkylphosphoryl, C
3
-C
20
-alkenylcarbonyl, C
3
-C
6
-alkynylcarbonyl, C
3
-C
20
-alkenylphosphoryl, C
3
-C
6
-alkynylphosphoryl, C
3
-C
20
-cycloalkyl, C
3
-C
20
-cycloalkylcarbonyl, C
3
-C
20
-cycloalkylphosphoryl, aryl, arylcarbonyl, arylphosphoryl, hetaryl, hetarylcarbonyl, hetarylphosphoryl, glycosyl, substituted or unsubstituted amino acids or peptides
R
2
a nucleofugic group
n 1 or 2.
Linkers according to the invention are linkers which contain a recognition site for a hydrolytic enzyme and are fragmented by reaction with the enzyme in such a way that the linker is completely eliminated from a synthesized product which is bound via the linker to the solid phase, i.e. no parts of the linker molecule remain in the synthesized product.
The linker is preferably eliminated from the product synthesized on the solid phase with elimination of CO
2
.
A recognition site for an enzyme means a linkage which can be cleaved by a hydrolytic enzyme. Examples of linkages which can be cleaved by hydrolytic enzymes are ester, amide, ether, phosphoric ester or glycoside linkages.
Suitable enzymes for cleaving the linker according to the invention under mild conditions are hydrolytic enzymes such as lipases, esterases, amidases, proteases, peptidases, phosphatases, phospholipases, peroxidases or glycosidases. Preferred enzymes are selected from the group of lipases, esterases, amidases, proteases or glycosidases, particularly preferably lipases, esterases or glycosidases.
Linkers according to the inve
Grether Uwe
Sauerbrei Bernd
Waldmann Herbert
BASF - Aktiengesellschaft
Keil & Weinkauf
Naff David M.
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