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Title:
OLIGOPHOSPHORAMIDATES
Document Type and Number:
WIPO Patent Application WO/2008/141799
Kind Code:
A1
Abstract:
The trivalent phosphorous atom of a compound is reacted with a reagent in such a manner that a stable phosphate mimetic is formed. Phosphoramidites with a phosphorous atom containing at least one hydroxyl residue which is provided with a protective group are reacted for this purpose with a free hydroxyl group: In the first synthesis cycle the hydroxyl group is linked to a solid support via a cleavable or non-cleavable linker. In further synthesis cycles the hydroxyl group is created by cleavage of the protective group from the growing oligomer. This results in formation of a phosphorous acid triester which is reacted with azides. Compounds of Formula (I) are produced.

Inventors:
HEINDL DIETER (DE)
SEIDEL CHRISTOPH (DE)
THUER WILMA (DE)
Application Number:
PCT/EP2008/004022
Publication Date:
November 27, 2008
Filing Date:
May 20, 2008
Export Citation:
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Assignee:
ROCHE DIAGNOSTICS GMBH (DE)
HOFFMANN LA ROCHE (CH)
HEINDL DIETER (DE)
SEIDEL CHRISTOPH (DE)
THUER WILMA (DE)
International Classes:
C07F9/24; C08G79/04
Domestic Patent References:
WO1996022297A11996-07-25
WO1997028168A11997-08-07
Foreign References:
EP0751948A11997-01-08
US3440222A1969-04-22
Other References:
PREOBRAZHENSKAYA N N: "Reactions of Phosphoramidic Acids", RUSSIAN CHEMICAL REVIEWS (USPEKHI KHIMII), INSTITUTE OF PHYSICS PUBLISHING, BRISTOL, GB, vol. 41, no. 1, 1972, pages 54 - 65, XP009092158, ISSN: 0036-021X
A. W. GARRISON, C. E. BOOZER: "The Acid-Catalyszed Hydrolysis of a Series of Phosphoramidates", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 90, no. 13, 1968, pages 3486 - 3496, XP002458590
BIRAN C. FROEHLER: "Deoxynucleoside H-Phosphonate Diester Intermediates in the Synthesis of Internucleotide Phosphate Analogues", TETRAHEDRON LETTERS, vol. 27, no. 46, 1986, pages 5575 - 5578, XP002458333
Attorney, Agent or Firm:
ROCHE DIAGNOSTICS GMBH (SimonPatent Department, Postfach 11 52 Penzberg, DE)
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Claims:
Patent Claims

1. A chemical compound according to Formula I,

(Formula I)

in which n is an integer denoting the number of monomers, and n is equal to or higher than 1,

in which Ace is an electron acceptor with a Hammett constant σ p which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60,

in which X is a moiety selected from the group consisting of 2-16 atoms branched alkyl, alkenyl, alkinyl, aryl, heteroaryl, cydoalkyl or cyclo- heteroalkyl structure, whereby the atom or atoms of X which are connected to phosphoramidate are sp 3 C atoms,

in which Rl is selected independently from R2, and Rl is directly or via a tether Tl attached to Ace,

in which R2 is selected independently from Rl, and R2 is directly or via a tether T2 to a C atom or (if present) N atom of X,

whereby Rl and R2 are moieties selected from the group consisting of

a hydrogen atom,

- a core moiety selected from the group consisting of

a linear or branched C1-C6 alkyl group,

a linear or branched C2-C6 alkenyl group,

a linear or branched C2-C6 alkinyl group, a 5-10-membered cycloalkyl group,

a 6- 10-membered aryl group,

■ a 5-10-membered heterocyclic group with 1-5 heteroatoms, whereby each heteroatom is independently selected from the group consisting of N, O, and S,

- a substituent attached to the core moiety, whereby the substituent is selected from the group consisting of

a hydrogen atom

a halogen atom

a carboxyl group, a formyl group,

an acyl group,

an aroyl group,

a hydroxyl group,

an amino group, an amido group,

a mercapto group,

a cyano group,

a nitro group,

an alkoxy group, ■ an alkoxycarbonyl group

an aryloxy group,

an aryloxycarbonyl group

■ a sulfhydryl group,

an aryl- or alkyl-sulfonyl group, " a phosphatyl group,

a guanidyl group,

a primary or secondary amido group

a detectable moiety

an aryl group, " a heteroaryl group,

and the tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety.

2. The chemical compound according to claim 1, characterized in that the electron acceptor selected from a group consisting of

alkyl-sulfonyl, aryl-sulfonyl, heteroaryl-sulfonyl - cycloalkyl-sulfonyl, an electron-deficient aromatic or heteroaromatic ring

3. The chemical compound according to claim 2, characterized in that the electron-deficient heteroaromatic ring is selected from a group consists of a six membered heterocycle with at least one alkylated N-atom in ortho- or para- position, said hetrocycle being selected from the group consisting of pyridinium, pyrimidinium and chinolinium.

4. The chemical compound according to claim 1, characterized in that X is a propyl residue and R2 is attached to the C2 atom of the propyl residue.

5. The chemical compound according to claim 1, characterized in that n is between 2 and 30.

6. Process for producing a compound according to claim 1, compring the steps

(a) providing a solid support to which is attached a hydroxyalkyl group via a cleavable or non-cleavable linker,

(b) reacting a first monomer according to Formula II

with the hydroxyl group of the solid support, to form a phosphoric acid triester,

in which X is selected independently for each monomer and X is a moiety selected from the group consisting of 2-16 atoms branched alkyl, alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo-heteroalkyl

structure, whereby the atom or atoms of X which are connected to phosphoramidite and to O-PG1 are sp 3 C atoms,

in which Rl is selected independently for each monomer and independently from R2, and Rl is directly or via a tether Tl attached to Ace,

in which R2 is selected independently for each monomer and independently from Rl, and R2 is directly or via a tether T2 attached to X,

whereby Rl and R2 are moieties selected from the group consisting of

- a hydrogen atom,

a core moiety selected from the group consisting of

• a linear or branched C1-C6 alkyl group,

• a linear or branched C2-C6 alkenyl group,

• a linear or branched C2-C6 alkinyl group, • a 5-10-membered cycloalkyl group,

• a 6-10-membered aryl group,

• a 5-10-membered heterocyclic group with 1-5 heteroatoms, whereby each heteroatom is independently selected from the group consisting of N, O, and S,

- a substituent attached to the core moiety, whereby the substituent is selected from the group consisting of

• a hydrogen atom

• a halogen atom

• a carboxyl group, • a formyl group,

• an acyl group,

• an aroyl group,

• a hydroxyl group,

• an amino group, • an amido group,

• a mercapto group,

• a cyano group,

• a nitro group,

• an alkoxy group,

• an alkoxycarbonyl group • an arγloxy group,

• an aryloxycarbonyl group

• a sulfhydryl group,

• an aryl- or alkyl-sulfonyl group,

• a phosphatyl group, • a guanidyl group,

• a primary or secondary amido group

• a detectable moiety

• an aryl group,

• a heteroaryl group,

and the tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety,

whereby reactive groups of Rl and R2 selected from the group consisting of carboxyl, formyl, hydroxyl, amino, amido, sulfhydryl, phosphatyl, imidazolyl, indolyl and guanidyl groups are protected by protective groups.

whereby the protective group PGl is selected from trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, trialkylsilyl, allyl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl

(MOX), nitroveratryl and NPPOC,

whereby the protective group PG2 is selected from betacyanoethyl, methyl, betanitroethyl and allyl,

and R3 and R4 are independent from each other and are selected from Cl - C6 alkyl, whereby R3 and R4 can be linked to each other by methylen or an O-atom to form a ring structure,

(c) reacting the phosphotriester of step (b) with Rl-Tl-Acc-azide (Formula III), whereby Ace is an electron acceptor with a Hammett constant σ p

which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60

(d) capping of non-reacted hydroxyl groups with an anhydride,

(e) cleaving of the temporary protective group PG 1 ,

(f) reacting the new deliberated hydroxyl group from step (e) with a second monomer according to Formula II,

(g) repeating steps (c) to (f) between 1 and 29 times,

(h) cleaving of PG2 and of the protective groups of the reactive substituents of Rl and R2.

7. The process according to claim 6, characterized in that the electron acceptor selected from a group consisting of

alkyl-sulfonyl, aryl-sulfonyl, heteroaryl-sulfonyl - cycloalkyl-sulfonyl, an electron-deficient aromatic or heteroaromatic ring

8. The process according to claim 7, characterized in that the electron-deficient heteroaromatic ring is selected from a group consists of a six membered heterocycle with at least one alkylated N-atom in ortho- or para- position, said hetrocycle being selected from the group consisting of pyridinium, pyrimidinium and chinolinium.

9. The process according to claim 6, characterized in that X is a propyl residue and R2 is attached to the C2 atom of the propyl residue.

10. Use of a compound according to any of the claims 1 to 5 or a compound obtainable by a process according to any of the claims 6 to 9 for modifying an enzymatic activity.

11. The use according to claim 10, characterized in that the enzymatic activity is enhanced.

12. The use according to claim 10, characterized in that the enzymatic activity is reduced.

13. The use according to any of the dims 10 to 12, characterized in that the enzymatic activity is selected from the group consisting of a protease, a phosphatase, a glycolytic hydrolase, and a polymerase.

14. A composition comprising (i) a compound according to any of the claims 1 to 5 or a compound obtainable by a process according to any of the claims 6 to 9, (ii) a polypeptide with enzymatic activity, (iii) a substrate capable of being converted by the enzymatic activity, and optionally (iv) a co-factor.

15. The composition according to claim 14, characterized in that the polypeptide has an enzymatic activity selected from the group consisting of a protease activity, a phosphatase activity, a glycolytic hydrolase activity, and a polymerase activity.

Description:

Oligophosphoramidates

The present invention is directed to oligomeric compounds consisting of monomeric units having a spacer segment covalently bound to a phosphoramidate moiety. Substituents are attached independently at both the spacer segment, and at the phosphoramidate moiety. The oligomers consist of either a random or a predefined sequence of units. A plurality of individual sequences can be created by varying the substituents of the incorporated monomers. The substituents differ from each another due to the choice of the preparator and the purpose of the prepared oligomers. The individual substituents can be selected from a broad range of chemical functionalities. Background of the Invention

EP 0 751 948 and Fathi, R., et al., J. Org. Chem. 61 (1996) 5600-5609 provides compounds and processes which are essentially based on the long known standard phosphoramidate modification of oligonucleotides (Vorob'ev, O., E., et al., Doklady Akademii Nauk SSSR 166 (1966) 95-98.).

Synthesis is based on the strategy of converting an H-Phosphonate with CC14 in the presence of an nucleophilic amine to the corresponding phosphoramidate (Froehler, B., C, Tetrahedron Letters 27 (1986) 5575-5578).

Acceptor-substituted amines could not be used since they are not nucleophilc and will not react with the dichlorophosphonate intermediate.

The P-N bond in standard phosphoramidates is known to be labile (Tomasz, J.;

Ludwig, J., Nucleosides & Nucleotides 3 (1984) 45-60), especially under slightly acid conditions.

Baschang, G., and Kvita, V., Angew. Chem. 85 (1973) 43-44 describe the reaction of a nucleotide phosphoric acid triester with azides such as methylsulfonyl azide to prepare tri-alkyl(aryl)imidophosphates which are, however, unstable and decompose.

Nielsen, J., and Caruthers, M., H., J. Am. Chem. Soc. 110 (1988) 6275-6276 describe the reaction of deoxynucleoside phosphites provided with a 2-cyano-l,l- dimethylethyl protective group in the presence of alkyl azide. Furthermore, the authors suggest that this principle is suitable for preparing nucleotides that are

modified on the phosphate residue without elucidating which types of modifications prepared with the aid of the disclosed method could have particular advantages. In particular the authors suggest the introduction of alkyl residues.

Therefore it was an object of the invention to provide compounds with a stabilized phosphoamidate linkage.

It was a further object of the invention to provide a new and simple preparative approach to generate a plurality of different individual oligomers consisting of a defined sequence of selected monomeric units. The monomeric units themselves are build of two parts, a phosphate containing part and a spacer part. The monomeric units themselves contain two groups Rl and R2 where Rl is connected to the phosphate containing part and R2 is connected to the spacer part of the monomeric unit.

Summary of the Invention

The objective was reached by a first embodiment of the invention, that is a chemical compound according to Formula I,

in which n is an integer denoting the number of monomers, and n is equal to or higher than 1, in which Ace is an electron acceptor with a Hammett constant σ p which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60, in which X is a moiety selected from the group consisting of 2-16 atoms branched alkyl, alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo- heteroalkyl structure, whereby the atom or atoms of X which are connected to phosphoramidate are sp 3 C atoms, in which Rl is selected independently from R2, and Rl is directly or via a tether Tl attached to Ace, in which R2 is selected independently from Rl, and R2 is directly or via a tether T2 to a C atom or (if present) N atom of X, whereby Rl and R2 are moieties selected from the group consisting of

- a hydrogen atom, a core moiety selected from the group consisting of

■ a linear or branched C1-C6 alkyl group,

a linear or branched C2-C6 alkenyl group,

■ a linear or branched C2-C6 alkinyl group,

■ a 5-10-membered cycloalkyl group, ■ a 6-10-membered aryl group,

■ a 5-10-membered heterocyclic group with 1-5 heteroatoms, whereby each heteroatom is independently selected from the group consisting of N, O, and S,

a substituent attached to the core moiety, whereby the substituent is selected from the group consisting of

a hydrogen atom

a halogen atom

a carboxyl group,

a formyl group, an acyl group,

an aroyl group,

a hydroxyl group,

an amino group,

an amido group, " a mercapto group,

a cyano group,

a nitro group,

an alkoxy group,

■ an alkoxycarbonyl group an aryloxy group,

an aryloxycarbonyl group

a sulfhydryl group,

■ an aryl- or alkyl-sulfonyl group,

a phosphatyl group, " a guanidyl group,

a primary or secondary amido group

a detectable moiety

an aryl group,

■ a heteroaryl group,

and the tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety.

Another embodiment of the invention is a process for producing a compound according to the invention, compring the steps

(a) providing a solid support to which is attached a hydroxyalkyl group via a cleavable or non-cleavable linker,

(b) reacting a first monomer according to Formula II with the hydroxyl group of the solid support, to form a phosphoric acid triester, in which X is selected independently for each monomer and X is a moiety selected from the group consisting of 2-16 atoms branched alkyl, alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo-heteroalkyl structure, whereby the atom or atoms of X which are connected to phosphoramidite and to O-PG1 are sp 3 C atoms, in which Rl is selected independently for each monomer and independently from R2, and Rl is directly or via a tether Tl attached to Ace, in which R2 is selected independently for each monomer and independently from Rl, and R2 is directly or via a tether T2 attached to X, whereby Rl and R2 are moieties selected from the group consisting of

a hydrogen atom, - a core moiety selected from the group consisting of

• a linear or branched C1-C6 alkyl group,

• a linear or branched C2-C6 alkenyl group,

• a linear or branched C2-C6 alkinyl group,

• a 5-10-membered cycloalkyl group, • a 6-10-membered aryl group,

• a 5-10-membered heterocyclic group with 1-5 heteroatoms, whereby each heteroatom is independently selected from the group consisting of N, O, and S,

- a substituent attached to the core moiety, whereby the substituent is selected from the group consisting of

• a hydrogen atom

• a halogen atom

• a carboxyl group,

• a formyl group, • an acyl group,

• an aroyl group,

• a hydroxyl group,

• an amino group,

• an amido group, • a mercapto group,

• a cyano group,

• a nitro group,

• an alkoxy group,

• an alkoxycarbonyl group • an aryloxy group,

• an aryloxycarbonyl group

• a sulfhydryl group,

• an aryl- or alkyl-sulfonyl group,

• a phosphatyl group, • a guanidyl group,

• a primary or secondary amido group

• a detectable moiety

• an aryl group,

• a heteroaryl group,

and the tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety,

whereby reactive groups of Rl and R2 selected from the group consisting of carboxyl, formyl, hydroxyl, amino, amido, sulfhydryl, phosphatyl, imidazolyl, indolyl and guanidyl groups are protected by protective groups. whereby the protective group PGl is selected from trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, trialkylsilyl, allyl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), nitroveratryl and NPPOC, whereby the protective group PG2 is selected from betacyanoethyl, methyl, betanitroethyl and allyl, and R3 and R4 are independent from each other and are selected from Cl - C6 alkyl, whereby R3 and R4 can be linked to each other by methylen or an O-atom to form a ring structure,

(c) reacting the phosphotriester of step (b) with Rl-Tl-Acc-azide (Formula III), whereby Ace is an electron acceptor with a Hammett constant σ p which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60

(d) capping of non-reacted hydroxyl groups with an anhydride,

(e) cleaving of the temporary protective group PG 1 , (f) reacting the new deliberated hydroxyl group from step (e) with a second monomer according to Formula II,

(g) repeating steps (c) to (f) between 1 and 29 times,

(h) cleaving of PG2 and of the protective groups of the reactive substituents of Rl and R2.

Further embodiments of the invention are oligophosphoramidates obtainable by the process according to the invention. Exemplary oligophosphoramidates are the oligophosphoramidates 1 to 11 as shown in Figures 6 to 16.

Detailed Description of the Invention

The central idea of the present invention was in this connection to start with a compound comprising a trivalent phosphorus atom and to react the trivalent phosphorous atom with a reagent in such a manner that a stable phosphate mimetic is formed as shown in Scheme 1 below. According to the invention the phosphoramidites according to Formula II with a phosphorus atom containing at

least one hydroxyl residue which is provided with a protective group are reacted for this purpose with a free hydroxyl group: In the first synthesis cycle the hydroxyl group is linked to a solid support via a cleavable or non cleavable linker. In further synthesis cycles the hydroxyl group is created by cleavage of the protective group PGl from the growing oligomer. This results in formation of a phosphorous acid triester which is reacted with the azides according to Formula III having the structure N = N = N - Acc-Tl-Rl in which Ace is an electron acceptor. Ace itself is substituted with a residue Rl and Rl can be selected from a wide range of organic substituents. Between Ace and Rl a tether moiety Tl may also be present. After completing all synthesis cycles, protective groups PG2 and further protecting groups which are attached if neccessary to Rl and R2 are cleaved off. This results in the formation of the compounds of Formula I with a pentavalent phosphorus atom to which a strongly electron-attracting electron acceptor group is covalently bound via an N-atom. This molecular assembly ensures that the compounds produced in this manner are resonance-stabilized and are therefore not susceptible to hydrolysis, in contrast to the phosphoramidate compounds known from the prior art.

linker- Support

Scheme 1

This idea underlying the invention can be applied to all processes in which a trivalent phosphorus is formed as an intermediate.

Phosphoramidites comprising the substituted spacer unit, to which a protected hydroxyl group (e.g. dimethoxytrityl protected) is attached are useful starting materials to introduce a monomeric unit during solid phase synthesis of a oligophosphoamidate. Phosphoramidites are activated by a weak acid, e.g. tetrazol or dicyanoimidazol, and than reacted with a hydroxylgroup of a monomeric unit which is already attached either via a cleavable or non cleavable linker to a solid support. This results in formation of phosphoric acid triesters with a trivalent phosphorus atom as intermediate products, whereas one of the phosphoric ester bonds is linked to the monomeric unit which was attached to the solid support, and the second bond is linked to the monomeric unit which is to be attached. The phosphorus atom is linked to a protected hydroxyl group such as for example to a beta-cyanoethyloxy group via the third ester bond. According to the invention this intermediate was reacted with an appropriate azide in the process of which the trivalent phosphorus atom is oxidized to a pentavalent atom by covalently linking -N-Acc-Rl to the phosphorus atom while releasing nitrogen. Oligophosphoamidate synthesis can then be subsequently continued by releasing the protective group (e.g. dimethoxytrityl) from the newly attached monomeric unit and reacting with a further phosphoramidite. After the desired product was synthesized the oligomer is cleaved from the solid support, e.g. by ammonia. During the cleavage process preferably all other protecting groups are removed, too. Stable polyphosphoramidates are obtained as end product which are modified in almost any manner on one or more phosphoramidate residues and on the spacer units linking the phosphoamidate moieties. If the first hydroxyl group is attached to the support via a noncleavable linker the protective groups are removed as described above but the oligophosphoamidate remains attached to the solid support. Notably, this is useful for preparation of arrays of oligophosphoamidates

Within the scope of the present invention some of the terms used are defined as follows:

The term protective group (or protecting group) denotes molecular assemblies which are connected to one or more functional groups of a molecule such that, as part of a multistep synthesis reaction, only one particular, non-protected functional group can react with the desired reaction partner. The skilled person differentiates between temporary and permanent protective groups. The first protects the side chains of the growing oligomer until the synthesis is finalized; the second protects the growing chain end of the oligomer and is removed before each prolongation

step. It is reintroduced with the incorporation of the next monomer. Generally, the use of one or more protective groups assures that oligomer synthesis proceeds in the desired way.

Examples of frequently used protective groups to protect hydroxyl groups are trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, trialkylsilyl, allyl, 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), beta- cyano-ethyl and others which are known to the skilled person. Protective groups for protecting amino groups are trifluoroacetyl, BOC, benzyloxycarbonyl, Fmoc and others. Other possible protective groups are summarized in standard text books (Greene, T., W., Protective groups in organic synthesis, John Wiley&Sons, Inc.

(1981) New York, Chichester, Brisbane, Toronto;Sonveaux, E., Methods in Molecular. Biology, Agrawal, S. (ed), Vol. 26, Protocols for Oligonucleotide Conjugates, Humana Press Inc., Totowa, NJ, 1994, Chapter 1).

The term spacer denotes the linkage between two phosphoamidate moieties. A spacer usually contains a characterizing substituent. The spacer is a trifunctional moiety where two of the arms are connected to the O-atoms of the adjacent phosphoamidates when an oligomer is formed. The third arm of the spacer contains a substituent from the group defined below. It may be a branched alkyl, heteroalkyl (an alkyl residue which additionally comprises one or more N atoms), alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo-heteroalkyl structure with three connectivities. Between the substituent and this branched structure a tether is optionally incorporated. If the spacer is located terminally, or in a monomer of the compound of the invention, one O-atom and/or the spacer is linked to a hydrogen, to a solid phase (optionally via a linker), to a detectable moiety or to phosphate or phosphoamidate.

Phosphoramidites are molecules containing a trivalent phosphorus atom which can be coupled to a hydroxyl group. Examples are beta-cyanoethyl-bis- diisopropylamino-phosphoramidite very well known from standard oligonucleotide synthesis. Especially useful are intermediates and phosphoramidites described in EP 1 186 613 (aminopropane spacer), Kawakami, J., et al, Chemistry

Letters 33 (2004) 1554-1555, (carboxy-ribose spacer) EP 1 431 297 (amino mannitol spacer) WO 03/104249 (pyrrolidin spacer) Korshun, V., A., et al, Synthetic Communications 26 (1996), 2531-2547 ( dialkylamin spacer). WO 97/43451 (1 amino 4,4 dimethyl cyclohexyl spacer), and Azam, A., T., M., Z., Chem. Commun. (2006), (3), 335-337. (carboxypropyl spacer).

The term "electron acceptor" encompasses atomic structures which have the tendency to attract free electron pairs. One measure of this is the Hammett constant. The present invention concerns in particular embodiments in which the Hammett constant σ p exceeds a certain value of 0.30, preferably 0.45 and particularly preferably 0.60 (Hansch, C, et al., Chem. Rev. 91 (1991) 165-195).

The electron acceptor must additionally be compatible with all chemical reactions in oligophorphoramidate synthesis i.e.

it should not be oxidized by iodine it must be inert towards acids i.e. dichloroacetic acid and trichloroacetic acid and it must be inert towards bases and in particular towards ammonia and it should not react with trivalent phosphoramidates.

Examples of electron acceptors which fulfil these conditions are:

SO 2 -alkyl, SO 2 -aryl, and electron-deficient aromatic and heteroaromatic rings like pyridyl, pyridylium, pyridazyl, tetrafluorophenyl, benzotriazyl. In addition these acceptors can also be bound to the nitrogen atom in a vinylogous or phenylogous manner. In addition to these acceptors also nitro- and cyano-acceptors can be bound to the nitrogen atom in a vinylogous or phenylogous manner.

The term "substituted" means that the structure that is referred to as being substituted contains another residue at any position provided this position is not defined in more detail. The term "optionally substituted" denotes that the structure referred to in this manner comprises embodiments with and without an additional residue.

The term "six-membered N + -heterocycle" encompasses N-heterocycles which are alkylated on an sp 2 nitrogen such that the overall charge of the heterocycle is positive. Examples of this are pyridinium, pyrimidinium and quinolinium. Such hetrocycles are known in the art to be electron deficient.

Compounds of the invention are shown by Formula I above. In Formula I, the bracketed portion is herein referred to as a monomeric unit. A monomeric unit is comprised of a spacer segment with a phosphoramidate attached thereto.

Compounds of the present invention are made up of at least 2 of these monomeric

units. Included in a monomeric unit is a phosphoramidate moiety that, in turn, is capable of bearing functional groups thereon. The phosphoramidate moiety is covalently bonded to a spacer segment which may also be capable of including a variety of functional groups covalently bonded thereto. Functional groups are covalently bonded directly to the backbone segment and the phosphoramidate, or via an optional tether group.

The spacer segment and phosphoramidate moiety serve as sites for connecting certain other groups that impart "functional" properties to the oligomeric compounds of the invention. By varying these functional groups - diversity is incorporated into the compounds of the invention.

The term tether (or linker) denotes a carbon chain having a length of 1 - 30 C- atoms or can also be a bisconnectable cyclic structure. A tether can also contain one or more internal heteroatom like nitrogen, oxygen, and/or sulphur and may thus comprise an amide or urea moeties. Tethers can also be branched, e.g. be dendritic. A tether interconnects a spacer or a phosphoramidite moiety with, e.g. a substituent, a functional group or a detectable unit which may optionally be protected by one or more protective groups. In the context of this invention, internal heteroatoms with the exception of a disulfide bond must be separated from each other by a minimum of two carbon atoms.

The groups Rl and R2 can be "reactive" or "non-reactive." By reactive, it is meant that they will interact with a target molecule in some manner (that need not but can be predefined). By nonreactive, it is meant that they are not designed to primarily interact with a target molecule, and in fact while they may interact with the target molecule, the primary purpose of the non-reactive moieties are to impart other properties to the molecule such as effecting uptake, distribution, metabolism or identification.

The functional groups are attached to the spacer segment and phosphoramidate moiety with or without intervening tethers. Tethers, as used in the context of this invention, are bivalent or polyvalent groups Such tethers can be used to position Rl and R2 in space with respect to the linear backbone or the phosphoramidate moiety of the oligomeric compound synthesized or to link Rl and/or R2 to the spacer or phosphoramidate moiety that themselves are not bindable to the parts of the monomeric unit.

Aryl groups according to the invention include but are not limited to substituted and unsubstituted aromatic hydrocarbyl groups such as phenyl and naphthyl groups. Aralkyl groups include but are not limited to groups having both aryl and alkyl functionality, such as benzyl and xylyl groups.

A number of functional groups can be introduced into compounds of the invention containing protective groups.

Solid supports useful for synthesis of compounds according to the invention include controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, R. H. et al., Nucleic Acids Research 19 (1991) 1527-1532), TentaGel Support - an aminopolyethyleneglycol derivatized support (see, e.g., Wright, P., et al.,

Tetrahedron Letters 34, (1993) 3373-3376) or Poros — a copolymer of polystyrene/divinylbenzene. Solid phase oligomer synthesis is in principle based on the well-known Merrifield synthesis and the Carruthers-Koster phosphoramidite method.

Another aspect of the invention is the use of a compound according to the invention for interacting with a target molecule. Likewise, another aspect of the invention is the use of a compound obtainable by a process according to the invention for interacting with a target molecule. According to the invention this interaction occurs in a solution, preferably in an aqueous solution.

As shown above, the compounds of the invention can easily be synthesized in a great variety. Thus, compounds can be synthesized which are capable of interacting with a target molecule. To this end, a combinatorial library of separately synthesized compounds according to the invention is contacted with a target molecule and interaction is assayed, thereby detecting interaction of one or more compounds and the target molecule. Following detection, a compound capable of interacting with the target molecule may undergo further refinement, preferably by exchanging one or more substituents such as Rl and R2 or by introducing modifications in Tl and T2, according to the invention. Thus, refinement can be used to fine-tune the interaction between the compound according to the invention and the target molecule.

The nature of the interaction can be manifold. Thus, as a result of the interaction the compound of the invention may physically interact with the target molecule.

Preferably, the interaction between the target molecule and the compound of the invention is specific.

Surprisingly, the interaction of a compound according to the invention with an enzyme as target molecule can be strong enough that the interaction can modify the enzymatic activity. Modification of an enzymatic activity can result from (i) interaction of the compound with the substrate that is targeted by the enzymatic activity, (ii) interaction of the compound with a co-factor of the enzyme, provided that such a co-factor is present in the enzymatic reaction, and (iii) interaction of the compound with the enzyme itself. Modification can occur characterized in that the enzymatic activity is enhanced or inhibited. Preferably, such modification is the effect of a specific interaction between the compound of the invention and the target.

Thus, the invention encompasses a method to modify the activity of an enzyme by contacting a mixture comprising an enzyme, a substrate and optionally a co-factor with a compound according to the invention under conditions permitting activity of the enzyme. In a preferred embodiment the activity of the enzyme is enhanced compared to the mixture without the compound according to the invention. In another preferred embodiment the activity of the enzyme is reduced compared to the mixture without the compound according to the invention.

Another aspect of the invention is a composition comprising (i) a compound according to the invention or a compound obtainable by a process according to the invention, (ii) a polypeptide with enzymatic activity, (iii) a substrate capable of being converted by the enzymatic activity. Optionally, the composition additionally comprises (iv) a co-factor. Preferably, one single compound according to the invention is present in the composition. Also preferred, the composition comprises one or more compounds according to the invention. The preferred concentration of the compound (or compounds) is in the range between 100 μM and 100 pM. Particularly preferred ranges include between 100 μM and 100 nM and between 100 nM and 100 pM.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figures

Figure 1 the structure of Formula I

Figure 2 the structure of Formula II

Figure 3 the structure of Formula III

Figure 4 the structure of Formula IV

Figure 5 Scheme 1

Figure 6 oligophosphoramidate 1

Figure 7 oligophosphoramidate 2

Figure 8 oligophosphoramidate 3

Figure 9 oligophosphoramidate 4

Figure 10 oligophosphoramidate 5

Figure 11 oligophosphoramidate 6

Figure 12 oligophosphoramidate 7

Figure 13 oligophosphoramidate 8

Figure 14 oligophosphoramidate 9

Figure 15 oligophosphoramidate 10

Figure 16 oligophosphoramidate 11

Figure 17 oligophosphoramidate 12

Figure 18 oligophosphoramidate 13

Figure 19 oligophosphoramidate 14

Figure 20 oligophosphoramidate 15

Figure 21 oligophosphoramidate 16

Figure 22 oligophosphoramidate 17

Figure 23 oligophosphoramidate 18

Figure 24 oligophosphoramidate 19

Figure 25 oligophosphoramidate 20

Examole 1

N-(l-[4,4 > -dimethoxytrityl]-serinolyl)-succinamic acid methyl ester (El) In this Example as well in those described below substances used in the reactions are disclosed in EP 1 186 613 and Digenis, G., A., et al., Journal of Medicinal Chemistry 29 (1986) 1468-1476.

2-Amino-3-(4,4 v -dimethoxytrityl]-propan-l,3-diol (1) (0.93g, 2.36 mmol) and succinic acid 2,5-dioxo-pyrrolidin-l-yl ester metyl ester (2) (0.6Og, 2,60 mmol)

were dissolved in 50 ml CH 2 Cl 2 and 990 μl (7 mmol) triethylamine was added. After stirring at r.t. for 5.5 h the solution was concentrated to dryness. The residue was purified by column chromatography on silica gel 60 (acetic acid ethyl ester). IH -NMR (dβ DMSO, δ) 7.76 (d, 1 H), 7.38 (d, 2 H), 7.32-7.18 (m, 7 H), 6.88 (d, 4 H), 4.61 (t, 1 H), 3,97 (m, 1 H), 3.73 (s, 6 H), 3.57 (s, 3 H), 3,46 (t, 2 H), 3.00 (m, 1

H), 2.91 (m, 1 H), 2.47 (m, 2 H), 2.40 (m, 2 H).

Example 2

N-(l-[4 ) 4 v -dimethoxytrityl]-3-[(2-cyanoethyl)-N,N-diisopropylphosphora midite]- serinolyl)-succinamic acid methyl ester (PAl) To a solution of compound El (100 mg, 0.19 mmol) in 3 ml CH 2 Cl 2 (i-Pr) 2 EtN

(103 μl, 0.6 mmol) and 2-cyanoethyl diisopropylphosphoramido chloridite (64 μl, 0.29 mmol) were added and stirred for 60 min. at r.t. (argon atmosphere). The solution was purified by flash column chromatography on silica gel (CH 2 Cl 2 /acetone 9:1). IH-NMR (CD3CN, 5) 7.47 (d, 2 H), 7.45-7.30 (m, 7 H), 6.89 (d, 4 H), 6.78 (t, 1 H), 4.19 (m, 1 H), 3.73 (s, 6 H), 3.71 (m, 3 H), 3.63 (s, 3 H),

3,61 (m, 2 H), 3.12 (m, 2 H), 2.60-2.44 (m, 5 H), 2,43 (m, 2 H) 2.91 (m, 1 H), 2.47 (m, 2H), 1.18 (m, 12 H). 31 P-NMR (CD3CN, δ) 148.65, 148.58.

Example 3

Synthesis of oligophosphoramidate 1

The oligophosphoramidate is synthesized in 1 μmol scale on ABI 394 synthesizer using the phophoramidite Uni-Link-Aminomodifier (BD Biosciences 5190-2) and PAl . As solid support is used a Phospholink-CPG (Roche Id 12239809). All chemicals for the standard protocol are from Proligo or ABI. The synthesis follows the standard protocol except for the oxidation. First oxidation occurs with 0.1 M 2- Azido-N-ethyl-pyridinium-tetrafluoroborate (RareChemicals) in acetonitrile 2 times for 30 min., second oxidation with 0.1 M 4-Acetamidobenzenesulfonyl azide (Fluka) in acetonitrile 2 times for 30 min.. The product is cleaved form the solid support and deprotected with 30 % aqueous NH 3 (4h 55°C).

Mass spectroscopy ( ESI-MS) calc: 781.76 found [M-H]: 782.91 Example 4

Further exemplary oligophosphoramidates

Using the chemistry of the invention, further structures according to the general structure of Formula I (Fig. 1) are synthesized. These include the structures shown in Figures 6 to 16. Example 5

Synthesis of N-(l-[4,4 v -dimethoxytrityl]-serinolyl)-3-phenyl propionamide) (E2)

2-Amino-3-(4,4 N -dimethoxytrityl]-propan-l,3-diol (1.60 g, 4.00 mmol) and

Hydrocinnamic acid N-hydroxysuccinimide ester (Tsou, Tai Li; Ho, Su Neng;

Chang, Li Ren, Zhonghua Yaoxue Zazhi (1993), 45(6), 563-572) (1.10 g, 4,50 mmol) were dissolved in 50 ml CH 2 Cl 2 and 1.7 ml (12 mmol) triethylamine was

added. After stirring at room temperature (r.t.) for 12 h the solution was concentrated to dryness. The residue was purified by column chromatography on silica gel 60 (acetic acid ethyl ester).

Mass spectroscopy ( ESI-MS) calc: 525.65 found [M-H]: 523.95 Example 6

Synthesis of N-(I- [4,4"-dimethoxytrityl]-serinolyl)-3-methylsulfanyl propionamide (E3)

2-Amino-3-(4,4 v -dimethoxytrityl]-propan-l,3-diol (0.2 g, 0.5 mmol) and 3- methylsulfanyl propionic acid N-hydroxysuccinimide ester (Iverson, Brent L.; Dervan, Peter B., Journal of the American Chemical Society (1987), 109(4), 1241-

1243) (0.19 g, 0.86 mmol) were dissolved in 5 ml CH 2 Cl 2 and 0.14 ml (1 mmol) triethylamine was added. After stirring at r.t. for 12 h the solution was concentrated to dryness. The residue was purified by column chromatography on silica gel 60 (acetic acid ethyl ester). ). IH-NMR (CD3CN, δ) 7.43 (d, 2 H), 7.32-7.2 (m, 7 H), 6.86 (d, 4 H), 6.46 (d, 1 H), 4.03 (m, 1 H), 3.76 (s, 6 H), 3.64-3.54 (m, 2 H), 3.12-

3.051 (m, 2 H), 2.69 (t, 2 H), 2.41 (t, 2 H), 2,07 (s, 3 H).

Example 7

Synthesis of 2-({2-[Bis-(4-methoxy-phenyl)-phenyl-methoxy]-ethyl}-phenyl- amino)-ethanol (E4) N-phenyldiethanolamine (Aldrich) (0.3 g, 1.6 mmol) was dissolved in 10 ml pyridin and 4,4"-Dimethoxytrityl chloride (Aldrich) (0.54 g, 1. 6 mmol) was added. After stirring at r.t. for 4 h the solution was concentrated to dryness. The residue was purified by column chromatography on silica gel 60 (acetic acid ethyl ester/ hexane 1:1). ). IH-NMR (D6, DMSO, 6) 7.43 (d, 2 H), 7.36-7.16 (m, 7 H), 7.09 (t, 2 H), 6.83 (d, 4 H), 6.61-6.50 (m, 3 H), 4.65 (t, 1 H), 3.72 (s, 6 H), 3.52-3.40 (m, 6

H), 3.12 (t, 2 H).

Example 8

Synthesis of N- ( 1 - [4,4 X -dimethoxytrityl] -3- [ (2-cyanoethyl)-N,N- diisopropylphosphoramidite]-serinolyl)-3-phenyl propionamide (PA2) To a solution of compound E2 (200 mg, 0.38 mmol) in 3 ml CH 2 Cl 2 (i-Pr) 2 EtN

(200 μl, 1.14 mmol) and 2-cyanoethyl diisopropylphosphoramido chloridite (127 μl, 0.57 mmol) were added and stirred for 60 min. at r.t. (argon atmosphere). The solution was purified by flash column chromatography on silica gel (CH 2 Cl 2 /acetone 9:1). IH-NMR (CD3CN, δ) 7.42 (d, 2 H), 7.32-7.14 (m, 12 H),

6.86 (d, 4 H), 6.23 (m, 1 H), 4.16 (m, 1 H), 3.73 (s, 6 H), 3.73-3.33 (m, 5 H), 3.09- 3.01 (m, 2 H), 2.88-2.82 (m, 2 H), 2.64-2.53 (m, 3 H), 2,43-2.38 (m, 2 H), 1.18 (m, 12 H). 31 P-NMR (CD3CN, δ) 147.46.

Example 9 Synthesis of N-(l-[4,4 v -dimethoxytrityl]-3-[(2-cyanoethyl)-N,N- diisopropylphosphoramidite]-serinolyl)-3-methylsulfanyl-prop ionamide (PA3)

To a solution of compound E3 (100 mg, 0.21 mmol) in 3 ml CH 2 Cl 2 (UPr) 2 EtN (108 μl, 0.62 mmol) and 2-cyanoethyl diisopropylphosphoramido chloridite (70 μl, 0.31 mmol) were added and stirred for 60 min. at r.t. (argon atmosphere). The solution was purified by flash column chromatography on silica gel

(CH 2 Cl 2 /acetone 85:15). IH-NMR (CD3CN, δ) 7.45 (d, 2 H), 7.31-7.20 (m, 7 H), 6.92 (m, 2 H), 6.87 (d, 4 H), 6.45 (m, 1 H), 4.20 (m, 1 H), 3.78 (s, 6 H), 3.78-3.50 (m, 6 H), 3.15-3.07 (m, 2 H), 2.87-2.83 (m, 2 H), 2.70-2.54 (m, 4 H), 2,43-2.38 (m, 2 H), 2.05 (s, 3 H) 1.18 (m, 12 H). 31 P-NMR (CD3CN, δ) 147.3. Example 10

Synthesis of PA4

To a solution of compound E4 (121 mg, 0.25 mmol) in 3 ml CH 2 Cl 2 (i-Pr) 2 EtN (131 μl, 0.75 mmol) and 2-cyanoethyl diisopropylphosphoramido chloridite (84 μl, 0.38 mmol) were added and stirred for 60 min. at r.t. (argon atmosphere). The solution was purified by flash column chromatography on silica gel (acetic acid ethyl ester/ hexane 1:1). IH-NMR (CD3CN, δ) 7.45 (d, 2 H), 7.30-7.11 (m, 9 H), 6.84 (d, 4 H), 6.70-6.61 (m, 3 H), 3.78 (s, 6 H), 3.78-3.22 (m, 10 H), 3.22 (t, 2 H), 2.59 (t, 2 H), 1.18 (m, 12 H). 31 P-NMR (CD3CN, δ) 148.5.

Example 11 Synthesis of Lysamine azide (Al)

To a solution of Sulforhodamine B (Fluka) (300 mg, 0.52 mmol) in 10 ml acetone sodium azide (40 mg, 0.62 mmol) in 0.5 ml H 2 O was added at 0 0 C and stirred for 12 h at r.t.. The solution was purified by column chromatography on silica gel (toluene/ acetic acid ethyl ester/ methanol 3:1:1).

Mass spectroscopy ( ESI-MS) calc: 583.69 found [M-H]: 581.82

Example 12

Synthesis of Oligophosphoramidates (12-20)

The Oligophosphoramidates (12-20) were synthesized in 1 mmol scale on an ABI 394 synthesizer using purchasable (GlenResearch), published (EP 1538221) or above described phosphoramidites. As solid support were used 3 V -Spacer-C3-CPG (GlenResearch) or Phospholink-CPG (GlenResearch) or 3 N -TFA-Aminomodifier- C7-CPG (Chem Genes). All chemicals for the standard protocol were from Proligo or ABI. The synthesis follows the standard protocol with a for 10 min. prolonged phosphoramidite coupling step. The oxidation of the trivalent phosphorus occurs with purchasable (Tolensulfonyl azide (VeZerf Laborsynthesen GmbH), 2-Azido-l- ethylpyridinium tetrafluoro borate, 4-Azido-l,2,6-trimethylpyridinium tetrafluoro borate (Rare Chemicals), 2-azido-5-nitro-pyrimidine (Toslab BB), 4- Acetamidobenzenesulfonyl azide (Fluka), KA3205 (Aurora)) or above described Azidocompounds (0.05 M in Acetonitril) 2 times for 30 min. instead of the standard procedure. The products were cleaved form the solid support and deprotected with 30 % NH 3 (2-4 h at r.t.).

Table 1: Mass spectroscopy data of oligophosphoramidate by ESI-MS

Example 13 Modification of beta-galactosidase activity by oligophosphoramidates

The experiment is performed using the enzyme beta-galactosidase in an ELISA immunoassay (Roche Cat. No. 11539 426 001) with ABTS as substrate. Each assay is performed according to the pack insert. Beta-galactosidase (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) is used as

standard. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the absorbance at 405 nm during the beta- galactosidase assay. For a given single oligophosphoramidate or a given mixture of two or more oligophosphoramidates each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s). Before the substrate is added, the oligophosphoramidate or the mixture of two or more oligophosphoramidates (preferably between 2 and 10 different oligophosphoramidate compounds) is added in an amount to have a final concentration in the range between 100 μM and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 μM and 100 pM). Changes in absorbance at 405 nm are monitored in comparison to the calibration curve.

Example 14 Modification of alkaline phosphatase activity by oligophosphoramidates

The experiment is performed using the enzyme alkaline phosphatase in an ELISA immunoassay with AttoPhos as substrate (Roche Cat. No. 11681982001). Each assay is performed according to the pack insert. Alkaline phosphatase (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) is used as standard. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the emission at 550 nm during the alkaline phosphatase assay. For a given single oligophosphoramidate or a given mixture of two or more oligophosphoramidates each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s). Before the substrate is added, the oligophosphoramidate or the mixture of two or more oligophosphoramidates is added in an amount to have a final concentration in the range between 100 μM and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 μM and 100 pM). Changes in emission at 550 nm are monitored in comparison to the calibration curve.

Example 15

Modification of protease activity by oligophosphoramidates

The experiment is performed with a protease (selected from pronase and trypsin) assay using resorufin-labeled Casein as substrate (Roche Cat. No. 11080733 001).

Each assay is performed according to the pack insert. Pronase or trypsin (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) are used as standards. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the emission at 584 nm during the protease assay. For a given single oligophosphoramidate or a given mixture of two or more oligophosphoramidates each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s). Before the substrate is added, the oligophosphoramidate or the mixture of two or more oligophosphoramidates is added in an amount to have a final concentration in the range between 100 μM and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 μM and 100 pM). Trypsin and pronase are assessed separately. Changes in emission at 584 nm are monitored in comparison to the respective calibration curve. Example 16

Modification of DNA polymerase activity by oligophosphoramidates

The effect of either a single oligophosphoramidate or a mixture of two or more oligophosphoramidates is analyzed in DNA amplification assay. PCR reactions in the presence or absence of 1 μM -100 μM of one or more oligophosphoramidates is performed in 50 μl reactions containing 25 ng, 10 ng, 5 ng, 1 ng and 0 ng of human genomic DNA, 30 mM Tris-HCl, pH 8.6, 1.5 mM MgCl 2 , 50 mM KCl, 0.2 mM dNTP's each, 0.4 μM primers (SEQ ID NO: 1 ATT AGA GAA CCA TGT TAA CAC TAC CG and SEQ ID NO: 2 GAG GTG AAT GAC CAC TGT TTA TTT TC ) and 2.5 units Taq DNA polymerase. The following cycle conditions are used: Initial denaturation for 4 min at 94°C and 35 cycles with 20 seconds denaturation at 94°C,

30 seconds annealing at 62°C, 60 seconds elongation at 72°C and a final elongation step of 7 min at 72°C. The influence on amplification is monitored by gel electrophoresis To this end, the products are separated on an agarose gel and visualized by ethidium bromide.