EP1966231A1

EP1966231A1 - Process for forming disulphide bridges

Info

Publication number: EP1966231A1
Application number: EP06841161A
Authority: EP
Inventors: Karsten Knorr; Marco Emgenbroich; Carsten BÜNGENER
Original assignee: AplaGen GmbH
Current assignee: AplaGen GmbH
Priority date: 2005-12-23
Filing date: 2006-12-23
Publication date: 2008-09-10
Also published as: WO2007076993A1; US20090081145A1; JP2009520739A

Abstract

The present invention relates to an improved process for forming disulphide bridges in substances which carry SH groups, more particularly peptides, for example by means of formation of intramolecular disulphide bridges, wherein a heterocyclic compound having at least one nitrogen atom (e.g. caffeine or a caffeine-like substance) is used to catalyse the reaction. Surprisingly it has emerged that the addition of the heterocyclic substance raises not only the yield but also the purity of product carrying disulphide bridges.

Description

AplaGen GmbH

Arnold-Sommerfeld-Ring 2, 52499 Baesweiler

Process for the formation of disulfide bridges

Various methods for forming disulfide bridges are known in the art. For example, in a standard method of cyclizing peptides by forming intramolecular disulfide bridges, it is known to use K ₃ [Fe (CN) e] as the oxidant. This reagent ensures clean cyclization products at high yields; Side reactions are avoided. In addition, cyclizations due to the influence of oxygen or iodine are still known. However, these methods have the disadvantage that they are either too slow or result in a large number of unwanted by-products. Another known method for the cyclization of peptides is the use of immobilized Ellmann's reagent (5,5; -dithiobis (2-nitrobenzoic acid)) as the oxidant. This method allows the complete oxidation of, for example, a linear peptide; Impurities caused by the reagent are avoided. This approach has recently been extended to cross-linked ethoxylate-acrylate-resin (CLEAR) supports. These are compatible with both organic and aqueous solvent mixtures.

Another method known in the art for the formation of disulfide bridges, for example for the cyclization of peptides, is the use of DMSO as the oxidizing agent. This process also results in complete oxidation of the linear peptide. However, this method has the disadvantage that the reaction proceeds very slowly and that the excess of DMSO must be removed before further processing, which is difficult with this organic solvent.

From the above, it follows that several methods for the formation of disulfide bridges, which in particular in the cyclization of peptides by training of intramolecular disulfide bridges is known. However, each of these methods has disadvantages in terms of yield, reaction rate or purity.

The present invention is therefore based on the object to provide an alternative method for generating disulfide bridges.

This object is achieved by a method for forming disulfide bridges, which is characterized in that the reaction is carried out in a liquid mixture containing at least one heterocyclic compound having at least one nitrogen atom in the ring.

Accordingly, claim 1 relates to a process for forming disulfide bridges, characterized in that the reaction is carried out in a liquid medium containing at least one compound which promotes the formation of disulfide bridges, said compound being selected from the group consisting of:

(a) a compound having in its structure a saturated or unsaturated six-membered heterocycle having at least one nitrogen atom, said heterocycle being adjacent to the nitrogen atom

Carbon atom has at least one hydroxyl group or an oxo group (= 0) according to the invention, wherein in the case of the presence of a hydroxy group, the heterocycle is unsaturated;

(b) a compound of the following general formula

in which the substituent A - is hydrogen, an optionally substituted alkyl radical, an optionally substituted aryl radical or a saturated or unsaturated heterocyclyl having 3 to 10 ring members and 1 to 3 heteroatoms, such as nitrogen, oxygen and / or sulfur, wherein the heterocyclyl unsubstituted or monosubstituted or polysubstituted by halogen, alkyl having 1 to 4 carbon atoms, cyano, nitro, Cycloalkyl having 3 to 6 carbon atoms, hydroxy, alkoxy having 1 to 4 carbon atoms and / or mercapto.

It has surprisingly been found that the above-defined compounds promote the formation of disulfide bridges and thus can act as a kind of catalyst in the reaction. It is therefore advantageous to add these compounds to the reaction mixture in order to promote the formation of disulfide bridges in the case of different SH group-carrying substances, in particular peptides and proteins.

According to one embodiment, the compound according to alternative (a) has the following basic structure:

wherein the heterocycle is saturated or unsaturated depending on the choice of the substituents R1 to R6 and may accordingly have one or more double bonds;

- V, W, X, Y and Z are either carbon atoms or nitrogen atoms, the heterocycle having a total of not more than three, preferably two, nitrogen atoms;

R1 represents either a hydroxy group or an oxo group according to the invention (= 0);

- R2 and R3, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is hydrogen or alkyl, an electron-withdrawing substituent, a functional group in particular a hydroxy group, an oxo group (= O) according to the invention, -CONH ₂ or an oxime (= N-OH) or R ₂ together with R _{3 represents} a five- or six-membered ring which may likewise have heteroatoms and optionally carries further substituents, or R2 and / or R3 are missing; - R4 and R5, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an electron-withdrawing substituent, a functional group such as in particular a hydroxy group, an oxo group (= 0) according to the invention, a carboxy group, -CONH ₂ or an oxime (= N-OH) or R4 together with R5 represents a five- or six-membered ring which may also have heteroatoms and optionally carries further substituents, R5 together with R6 represents a five- or six-membered ring which may also have heteroatoms and optionally further substituents carries or R4 and / or R5 are missing;

- R6 represents hydrogen or an optionally substituted alkyl or aryl radical, or R6 together with R5 represents a five- or six-membered ring, which may also have heteroatoms and optionally carries further substituents, or R6 is missing.

The abovementioned substituents R 2 to R 6 may be absent if a nitrogen atom having a double bond is present at the linking position in the ring (see, for example, the compounds 2,6-dihydroxypyridine hydrochloride; uracil-6-carboxylic acid, 4,6 -Dihydroxypyrimidin).

A central feature of the substances according to the invention according to alternative (a) is the presence of the 6-membered, nitrogen-containing heterocycle and the hydroxy or oxo group (= 0) according to the invention on the adjacent carbon atom. "Oxo group" in the context of the invention means that the respective substituent forms an oxo group with the ring atom and, correspondingly, an oxygen atom is bonded to the ring via a double bond:

Extensive experiments have shown that compounds without such a functional group (hydroxyl group, oxo group = 0) regularly did not have the ability to promote the formation of disulfide bridges (for example, aminopyrazine, 2,4-diaminopyrimidine, melamine, pyrazinecarboxylic acid and pyrazinamide) ,

As is clear from the definition of the substituents, there are compounds having only one nitrogen atom in the heterocycle and having the advantageous effects of promoting the formation of the disulfide bonds. An example of one

Compound with only one nitrogen atom in the heterocycle and one oxo group (= 0) according to the invention on the adjacent carbon atom is the compound N-methyl-2-pyridone:

Another example of a compound having only one nitrogen atom in the heterocycle and having one hydroxyl group on the adjacent carbon atom is the compound 2,6-dihydroxy-pyridine hydrochloride:

As stated in the claim, the heterocycle is unsaturated in the case of the presence of hydroxyl groups. Experiments have shown that in this case the presence of at least one double bond is obviously important for the catalytic effect. Without wishing to be bound by it, it is speculated that this may be due to the fact that compounds of this structure can tautomerize. Tautomers are structural isomers that differ only in the position of a group (eg, hydrogen) and in the position of a double bond. In the case of the hydroxy and the oxo group (= 0) one also speaks of keto-enol tautomerism. It has proved to be advantageous if the aromatically present compounds, such as the 2,6-dihydroxy-pyridine hydrochloride can tautomerize. This is especially true for the enol forms, which may preferentially tautomerize toward the keto form. Tautomeric (isomeric) forms of the substances shown are therefore included according to the invention.

Preferably, the six-membered heterocycle has two nitrogen atoms which may be positioned differently. Examples of active compounds of this structure are uracil-6-carboxylic acid, 2,4-dihydroxy-6-methylpyrimidine, 2,4-dimethyl-6-hydroxypyrimidine, 2-isopropyl-6-methyl-4-pyrimidinol, 4,6-dihydroxy 2-methylpyrimidine, 4,6-dihydroxypyrimidine, 1, 2-dihydro-3,6-pyridazinedione:

An example of compounds in which R5 and R6 together form a ring structure is 7-hydroxy-5-methyl [1,2,4] triazolo [1,5-a] pyrimidine:

According to a further embodiment, the compound has the following substructure:

wherein the heterocycle is saturated or unsaturated depending on the choice of the substituents R2, R3, R4 and R6 and may accordingly have one or more double bonds; in which

- R2 and R3, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is hydrogen or alkyl, an electron-withdrawing substituent, a functional group in particular a hydroxy group, an oxo group (= O) according to the invention, -CONH ₂ or an oxime (= N-OH) or R ₂ together with R _{3 represents} a five- or six-membered ring which may likewise have heteroatoms and optionally carries further substituents, or R2 and / or R3 are missing; - R4 is hydrogen or an optionally substituted alkyl or aryl radical or R4 is absent;

- R6 is hydrogen or an optionally substituted alkyl or aryl radical or R6 is missing.

Active examples of this embodiment are, for example, barbituric acid, alloxan monohydrate and violuric acid

Further examples of this embodiment are uracil derivatives of the following general formula:

wherein R4 and R6 are independently hydrogen or an optionally substituted alkyl or aryl radical, preferably hydrogen or a branched or unbranched C1 to C10 alkyl radical, particularly preferably a C1 to C4 alkyl radical or hydrogen.

Examples of good activity are uracil and 1-methyl-uracil:

According to a further embodiment, the compound which promotes the formation of disulfide bonds is purine derivatives whose skeleton corresponds to the following general formula

wherein the five-membered ring is unsaturated and correspondingly has double bonds and wherein

R4 and R6, each independently, are hydrogen, an optionally substituted alkyl group or an optionally substituted aryl group; preferably hydrogen, an optionally substituted Ci- to Cio-alkyl radical or an optionally substituted C ₆ - or Cio-aryl radical; particularly preferably hydrogen, an optionally substituted C ₁ to C ₆ alkyl radical or an optionally substituted C ₆ aryl radical; in particular represent hydrogen or an optionally substituted C ₁ to C ₃ alkyl radical;

- R7, R8 and R9, each independently of one another, represent hydrogen, an optionally substituted alkyl radical, an optionally substituted aryl radical or an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is hydrogen or alkyl or a functional Stand group; preferably hydrogen, an optionally d- substituted by Ci _O-alkyl radical, an optionally substituted C ₆ - or Cio-aryl or an optionally substituted radical - {CH ₂₎ _n -COOX where n = 1 to 10 and X is hydrogen or C _r to C ₈ alkyl; particularly preferably hydrogen, an optionally substituted d- ₆ alkyl to C, an optionally substituted C ₆ aryl group or an optionally substituted - (CH ₂₎ _n -COOX where n = 1 to 6 and X is hydrogen or C _r to C _6- alkyl; in particular hydrogen, an optionally substituted C ₁ to C ₃ -alkyl radical or an optionally substituted radical - (CH ₂ ) _n -COOX with n = 1 to 4 and X is hydrogen or C ₁ - to C ₃ -alkyl.

As stated, the cyclic compound can thus be based on a purestan skeleton. The purine skeleton can be regarded as a condensed ring system composed of the two heterocycles pyrimidine and imidazole. Its systematic IUPAC name is 7H-imidazole [4,5-d] pyrimidine. The 7H-purine is in a tautomeric equilibrium with its isomer, the 9H-PuHn, and in the context of the present invention, compounds based on both tautomeric forms are also considered:

7H purine 9H purine

Depending on the choice of substituents on the pyrimidine ring this may also have fewer double bonds.

In a very particularly preferred variant of the present invention, at least one radical R4, R6, R7 and R9 is an alkyl group and at least one, preferably two of the radicals R4, R6, R7 and R9 represent hydrogen or a C ₁ to C ₃ alkyl group, preferably Methyl.

Specific examples of these heterocyclic compounds based on purine are 3-methylxanthine, theobromine, theophylline, caffeine, isocaffeine, xanthine, theophylline-7-acetic acid, theophylline-8-butyric acid and 3-isobutyl-1-methylxanthine:

As these selected examples show, tautomeric compounds based on the 9H-purine backbone instead of the 7H-purine backbone shown above are also included. In another embodiment, R2 and R3 together form an optionally substituted six-membered ring optionally having at least one heteroatom. This embodiment is preferably compounds in which an aromatic has been fused to the basic heterocycle. This prefers a bridge of two carbon atoms. Examples in which a 5-membered ring has been condensed (for example imidazole) have already been explained above (compounds based on the purine skeleton). Further examples of fused ring structures are pyrazine and quinoxaline.

An example of a compound having a benzene ring as a fused 6-membered ring is 1, 2,3-benzotriazine-4 (3H) -one:

According to an advantageous embodiment, the compound has the following basic structure:

wherein the rings may be unsaturated depending on the choice of the substituents and may carry one or more double bonds, and accordingly

R1 represents either a hydroxy group or an oxo group of the invention (= 0);

R4 and R6, each independently of the other, are or are absent for hydrogen, an optionally substituted alkyl radical or an optionally substituted aryl radical; preferably hydrogen, an optionally substituted branched or unbranched C _r to C ₁₀ alkyl radical or an optionally substituted C ₆ - or C ₁₀ -

Aryl radical; particularly preferably represents hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical or a given if substituted C ₆ aryl radical; in particular hydrogen or an optionally substituted C ₁ to C ₃ alkyl radical;

R5 is hydrogen, an optionally substituted alkyl or aryl radical, an electron-withdrawing substituent, a functional group such as in particular a hydroxy group or an oxo group according to the invention (= 0) or R5 is absent;

R10 and R13, each independently of one another, are hydrogen, an optionally substituted alkyl radical or an optionally substituted aryl radical, or R10 and / or R13 are absent; are preferably hydrogen, an optionally substituted branched or unbranched C _{1 to} C ₁ ₀ alkyl radical or an optionally substituted C ₆ or C 10 aryl radical; particularly preferably hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical or an optionally substituted C ₆ -aryl radical; in particular represent hydrogen or a C ₁ to C ₆ -alkyl radical substituted by at least one hydroxyl group;

R 11 and R 12 are each independently of one another hydrogen, an optionally substituted alkyl or aryl radical, an electron-withdrawing substituent, a functional group such as a hydroxy group, an oxo group (= O) according to the invention, a carboxy group, -CONH ₂ or an oxime (= N) OH) or R11 and R12 together form a five- or six-membered ring, which may optionally carry further heteroatoms and substituents.

Selected examples of compounds covered by this formula are, for example, (-) - riboflavin, lumazine and alloxazine

It has proved to be advantageous for the effect that the compounds according to the invention according to the alternative (a) do not carry any exocyclic amino groups. The compounds to be used according to the invention therefore preferably have no exocyclic amino groups.

The above-mentioned substituents R2, R3, R4 and R5 may also independently represent hydrogen, an optionally substituted branched or unbranched C ₁ - to C ₁₀ -alkyl radical or an optionally substituted C ₆ - or C ₀ -

aryl; preferably hydrogen, an optionally substituted branched or unbranched C ₁ - to C ₆ -alkyl radical or an optionally substituted C ₆ -aryl radical; in particular hydrogen or an optionally substituted C ₁ to C ₃ -alkyl radical, in particular methyl radical.

R 6 is preferably hydrogen or an alkyl radical, C ₁ to C ₈ , preferably C ₁ to C ₄ , particularly preferably hydrogen or a methyl group.

Electron-withdrawing groups or atoms, which may also be used herein as substituents on the heterocycle (see above), are, for example, electron-withdrawing groups or atoms which as substituents lower the electron density on a corresponding aromatic heterocyclic ring (also referred to as deactivating groups). Electron-withdrawing groups have an (-) - M and / or a (-) - I effect. The resonance effect (M effect, mesomeric effect) generally only works when the group is directly attached to the unsaturated heterocyclic system. It acts via π-electrons, in contrast to the field effect (I-effect, inductive effect), which acts through space, through solvation molecules or preferably via σ bonds of a system.

An electron-withdrawing effect can be either inductive (i.e., by the so-called (-) I effect) and / or mesomerically (i.e., by the so-called (-) - M effect). The classification of aromatic substituents in substituents with (+) - l and (-) - l effect and with (+) - M effect and (-) - M effect is known in the art per se. For further details, reference is made to Beyer / Walter, "Lehrbuch der Organischen Chemie", 1998, 23rd revised and updated edition, pages 515 to 518, the disclosure of which is included in the present invention.

Some non-limiting examples of (-) M effect groups are -SO ₂ -, -SO ₂ O-, -00-, -COO-, -CONH-, -CONR-, -SOR-, -CN , -NO ₂ , -CHO, -CO-, -COSH, -COS ^" , -SO ₃ H and the oxo group according to the invention (= 0). As can be seen from this, the terms" electron-withdrawing group "and" functional group "can be used. Corresponding groups are examples of useful substituents. According to a further embodiment, the compound according to alternative (b) has a substituent A which

is hydrogen, an optionally substituted C ₁ - to C _O alkyl group, an optionally substituted C ₆ - or Cio-aryl or saturated or unsaturated heterocyclyl having 3 to 10 ring members and 1 heteroatom such as nitrogen, oxygen and / or sulfur, where the heterocyclyl is unsubstituted or mono- or polysubstituted by halogen, alkyl having 1 to 4 carbon atoms, cyano, nitro, cycloalkyl having 3 to 6 carbon atoms, hydroxy, alkoxy having 1 to 4

Carbon atoms and / or mercapto; preferably represents hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical, an optionally substituted C ₆ -aryl radical or saturated heterocyclyl having 5 or 6 ring members and 1 heteroatom, such as nitrogen, oxygen and / or sulfur, wherein the heterocyclyl is unsubstituted or monosubstituted or polysubstituted by halogen, alkyl having 1 to 4 carbon atoms, cyano, nitro, cycloalkyl having 3 to 6 carbon atoms, hydroxy, alkoxy having 1 to 4 carbon atoms and / or mercapto; in particular hydrogen, an optionally substituted C ₁ to C ₃

Alkyl radical or saturated heterocyclyl having 5 or 6 ring members and 1 heteroatom, such as nitrogen, oxygen and / or sulfur, wherein the heterocyclyl is unsubstituted; stands.

Specific examples of these pyrimidine derivatives are the following compounds:

Aminexil

minoxidil In order to facilitate the binding of the substance to the carrier, functional groups on the heterocyclic compound to be used according to the invention are helpful. Therefore, other, not explicitly mentioned before derivatives of heterocyclic compounds can be used according to the invention.

The heterocyclic compounds described above can be used both in pure form and as mixtures of various possible isomeric forms, in particular of stereoisomers, such as E and Z, threo and erythro, and optical isomers, such as R and S isomers or atropisomers and of tautomers. The invention includes both the pure isomers and their mixtures.

Depending on the nature of the substituents defined above, the heterocyclic compounds have acidic or basic properties and can form salts, optionally also internal salts. If the compounds of the formula (I) bear hydroxy, carboxy or other groups which induce acidic properties, these compounds can be reacted with bases to form salts. Suitable bases are, for example, hydroxides, carbonates, bicarbonates of the alkali and alkaline earth metals, in particular those of sodium, potassium, magnesium and calcium, furthermore ammonia, primary, secondary and tertiary amines having (C _r C ₄ -) - alkyl radicals and mono-, di and trialkanolamines of (C 1 -C ₄ ) -alkanols. If the compounds of the formula (I) bear amino, alkylamino or other basic-property-inducing groups, these compounds can be reacted with acids to form salts. Suitable acids are, for example, mineral acids, such as hydrochloric, sulfuric and phosphoric acids, organic acids, such as acetic acid or oxalic acid, and acidic salts, such as NaHSO ₄ and KHSO ₄ . The salts thus obtainable are also usable.

Further preferred groups of the above compounds are discussed below.

Examples of heterocyclic compounds which can be used according to the invention for promoting the formation of the disulfide bridges can also be described as follows:

The radicals R ₁ ', R ₂ ' and R ₃ 'are either the same or different; However, at least one of the radicals represents an alkyl group. It is understood that tautomers, in which Among other things, the double bond migrates, also be captured by the above formula. Corresponding structural isomers are therefore also encompassed by this formula. As has been stated, these compounds are particularly suitable for promoting the formation of disulfide bridges in amino acid-containing substances, in particular in peptides and proteins.

At least one, preferably two of the radicals R ₁ ', R ₂ ' and R ₃ ', which are either identical or different, preferably represents either hydrogen or a Ci to C ₅ alkyl group. In particular short-chain alkyl groups having 1 to 3 carbon atoms, in particular the Methyl group, have proved to be beneficial. Further, at least one of the radicals may include a functional group. Furthermore, it is also possible that residues R ₄ '(on the nitrogen) and R ₅ ' (on the carbon between the nitrogen atoms) are present at the remaining positions on the heterocyclic 5-membered ring (especially in the tautomeric forms). These are customizable, preferably organic radicals. According to one embodiment, they are functional groups which allow the attachment of the substance to, for example, a carrier. This variant will be described in detail below.

Particularly suitable substances for use with the method according to the invention are, as already explained, caffeine and caffeine-like substances such as, for example, theobromine and theophylline.

It has surprisingly been found that in particular peptides and proteins, in particular peptides having an amino acid length between 5 and 100, preferably 10 and 50, more preferably between 15 to 40 amino acids in water even at higher peptide concentration at room temperature by the method according to the invention cyclize by formation of intramolecular disulfide bridges. The method is therefore particularly suitable for the formation of intramolecular disulfide bridges and therefore in particular for the cyclization of peptides. Furthermore, polypeptides and proteins can be cyclized with the appropriate method. Furthermore, it is also possible to form disulfide bridges with differently structured substances carrying SH groups. The formation of the disulfide bridges, for example, in a cyclization, takes place due to the addition of the heterocyclic compound of the invention such as caffeine or a caffeine-like substance (see above formulas) in some peptides almost quantitatively. However, this is not mandatory. Surprisingly, it is not necessary (but possible) to add an oxidizing agent to accelerate the reaction, since the atmospheric oxygen in the presence of the above-characterized substance for the formation of disulfide bridges is sufficient.

Experimental results have further shown that in the method according to the invention, the reaction rate may increase in the course of the reaction. The positive Influencing by the inventive addition of the above-characterized substance is surprising in that it is mostly - in contrast to the substances used in the prior art DMSO or iodine - not oxidizing agent. In the method according to the invention, the atmospheric oxygen is sufficient for the oxidation, wherein the reaction rate increases in an advantageous manner due to the addition of the heterocyclic compound according to the invention. Increasing the reaction rate might also suggest an autocatalytic mechanism, possibly through the cyclized product.

Advantageously, it has been shown that despite the increase in the reaction rate and even when using high peptide concentrations, often no or only little oligomerization occur. The formation of intermolecular disulfide bridges, which is undesirable in the case of cyclization of a peptide, was therefore not observed. However, the peptide concentration to be used depends on the particular peptide used and should therefore be optimized for each peptide.

The process according to the invention can advantageously be carried out at room temperature.

The apparently autocatalytic course of the reaction occurs both in unbuffered and in buffered solutions (for example phosphate buffer, pH 6 - 9). However, it has been shown that the reaction rate can be significantly increased when the pH is lowered. It is therefore advantageous to adjust the pH at <= 7, preferably in a pH range of about 4 or 5 to 6.5, more preferably a pH value around 6 (5.5 to 6.5).

The amount of substance added to the reaction mixture to promote the formation of the particular intramolecular disulfide bridges will vary depending on the compound and the substance to which disulfide bridges are to be formed. As a rule, small catalytic amounts are sufficient. The amount to be employed is preferably at least about 0.0001 mg / ml, more preferably in a range of about 0.0001, 0.001 or 0.01 to 20 mg / ml, 0.001 or 0.01 to 15 mg / ml, 0.001 or 0.01 to 10 mg / ml, 0.001 or 0.01 to 5 mg / ml, preferably 0.001 or 0.01 to 1 mg / ml, and more preferably in a range of 0.03 to 0.5 mg / ml. The amounts vary depending on the selected substance (eg caffeine or caffeine-like substance) and the peptide or protein to be treated and should therefore be individually optimized in each case. A particularly suitable concentration range for peptides with a length of about 15 to 25 amino acids (in particular in the case of EPO-mimetic peptides) is 0.05 to 0.3 mg / ml, more preferably 0.075 to 0.15 mg / ml. However, the amounts vary depending on the peptide and can also be significantly higher; the amounts should therefore preferably be optimized for the particular peptide. The reaction rate can be further accelerated if an additional oxidizing agent is added to the reaction mixture. For example, glutathione in oxidized form (GSSG) may be mentioned.

Surprisingly, it has been shown that the cyclization can be carried out effectively even at high peptide concentrations, without unwanted Oligomeri- sations occur. High peptide concentrations are therefore rather unproblematic in the process according to the invention in the case of many peptides. Rather, it has later been shown with some peptides that according to the process of the invention, the cyclization reaction proceeds even better at high peptide concentrations. Depending on the peptide, suitable peptide concentrations are about 0.05 or 0.1 or 0.5 to 5 mg / ml, concentrations in a range from 0.7 to 1.5 mg / ml are preferred. The exact concentration of course depends on the particular peptide, its length and its amino acid composition and varies accordingly. The information given is therefore not restrictive. For EPO-mimetic peptides, it has proved to be particularly advantageous to use a concentration of about 0.7 to 1 mg / ml. They can be cyclized particularly effectively by the addition of caffeine.

Usually, a disulfide bridge is formed in a peptide or protein between two cysteines. However, according to the invention, the disulfide bridge can also be formed between other natural and non-natural amino acids, provided that they have appropriate groups which are suitable for forming a disulfide bridge (-S-S-). Thiolysin, homocysteine and other cysteine derivatives are to be mentioned as examples of suitable amino acids in addition to cysteine. However, the term disulfide bridge is not to be equated with the term cysteine bridge, but includes the formation of corresponding -S-S bonds between any natural or non-natural SH-containing amino acids or other SH-containing compounds. With the method according to the invention can therefore also disulfide bridges in other SH-containing compounds, in particular polymers, train. Of course, a plurality of disulfide bridges can also be formed with the method according to the invention.

The present process can be used particularly advantageously for the cyclization of EPO-mimetic peptides (see, for example, WO 96/40479). Novel EPO mimetic peptides are described in PCT / EP2005 / 012075 (WO 2006/050959), the disclosure content of which with respect to the peptides is hereby made the subject of this application in its entirety. As described in detail in PCT / EP2005 / 012075 (WO 2006/050959), these novel EPO mimetic peptides have no proline in position 10 of the EPO mimetic consensus motif (with regard to the Numbering is referred to Johnson et al, 1997). Rather, the proline is replaced by a non-conservative amino acid, in particular a basic amino acid, in particular lysine.

The peptides described in PCT / EP2005 / 012075 (WO 2006/050959) preferably have the following consensus:

X6X7X8X9X10X11 Xi 2X13X14X15

wherein each amino acid represents a natural or non-natural amino acid and

X ₆ is C, A, E, α-amino-γ-bromobutyric acid or homocysteine (Hoc);

X ₇ is R, H, L, W or Y or S;

X ₈ is M, F, I, homoserine methyl ether (Hsm) or norisoloisine;

X ₉ is G or a conservative substitution of G; X ₁₀ is a non-conservative substitution of proline; or X ₉ and X ₁₀ are substituted by a single amino acid;

X ₁₁ may be any amino acid;

X ₁₂ is T or A;

X ₁₃ is W, 1-nal, 2-nal, A or F; X ₁₄ is D, E, I, L or V;

X ₁₅ is C, A, K, α-amino-γ-bromobutyric acid or homocysteine (Hoc) with either X ₆ or X ₁₅ C or Hoc.

EPO mimetic peptides show a particularly good activity in cyclized form. Usually, therefore, two peptide monomers (the monomers correspond to binding domains) are each cyclized with an EPO mimetic consensus and linked to a dimer, since binding to the EPO receptor in this form is most effective. The EPO mimetic monomers have an average of 10 to 25 amino acids. Preferably, as described in PCT / EP2005 / 012075 (WO 2006/050959), they are synthesized as continuous dimers (bivalent peptides) to avoid separate dimerization steps.

Various methods for the formation of intramolecular disulfide bridges for the cyclization of EPO-mimetic or TPO-mimeti- see peptides are known in the art. At the heart of the known teachings is the oxidation of the cysteine residues (or corresponding other SH group-containing amino acids) in the EPO mimetic consensus. As a typical oxidizing agent DMSO has been used, but this has the disadvantages described above. Cyclization by the process according to the invention has some decisive advantages over the processes known in the prior art. Thus, better yields and greater purity of the product are achieved than with the method known in the art. Another decisive advantage of the method according to the invention is the simple separability of the cyclization reagent according to the invention from the reaction product by a simple HPLC. According to a further variant, the heterocyclic compound (for example caffeine) can be separated off by liquid-liquid extraction. For example, caffeine can be separated from an aqueous peptide solution by repeated extraction with dichloromethane. In the cyclization of longer peptides, the use of size exclusion chromatography (SEC) is also possible. The purification is therefore considerably simplified.

Depending on the SH group-containing substance or peptide and the reaction conditions used, the reaction time can be reduced to less than eight hours (for example by lowering the pH, choosing an additional oxidizing agent). Usually, the reaction time is <= twenty-four hours, preferably less than twenty hours, more preferably less than fifteen hours, and most preferably between five and ten hours.

In addition to the mentioned EPO mimetic peptides, however, other peptides were also successfully cyclized by the method according to the invention. For example, on the peptide derived from oxytocin which, in contrast to oxytocin, has a carboxylic acid on the C-term instead of an amide:

H-CYIQNCPLG-OH which is also cyclized by formation of an intramolecular cysteine bridge.

As mentioned, the intramolecular disulfide bridge is preferably formed between two amino acids. These may be natural or unnatural, the only prerequisite is the ability to form a disulfide bond by reacting the SH group. Cysteine is arguably the best-known disulfide bridge-forming amino acid, which is also used in nature predominantly for the formation of disulfide bridges. Disulfide bridges occur in nature especially in the formation of intra- and intermolecular disulfide bridges. Thus, for example, they effect the cohesion between the individual polypeptide chains of proteins (for example insulin) in the form of intermolecular disulfide bridges and regularly stabilize the conformation within a protein by forming intramolecular disulfide bridges.

The keratin of wool and hair contains, for example, more than 10% cysteine, which is why there are also many disulfide bridges. Are these disulfide bridges broken (For example, by lyes, light, heating, etc.), the tensile strength of the fibers decreases sharply. The method according to the invention can therefore also be used to form disulfide bridges in fibers (natural and synthetic fibers). The same applies to the treatment of hair, in which disulfide bridges are also of great importance for structural strength. The method according to the invention can therefore also be used to form disulfide bridges in hair, which also opens up a field of cosmetic application (for example shampoos, reagents for forming perms, etc.). Thus, the method according to the invention can be used, for example, as means for closing the disulfide bridges in the context of a permanent wave treatment. For this purpose, it is particularly advantageous if, in addition to the substance characterized in more detail above, an oxidizing agent is added to promote disulfide bridge formation. It has been found that the reaction rate can be significantly accelerated and the disulfide bridges are closed correspondingly faster. This has the consequence that when applying the method according to the invention for hair, the contact time and thus the duration of the treatment for the customer is shortened in an advantageous manner. A particularly suitable oxidizing agent is oxidized glutathione (GSSG). The resulting improvement in the closure of disulfide bridges in both quantitative and temporal terms is clearly described in the experimental examples.

Accordingly, the present invention also relates to the use of the previously described heterocyclic compounds in cosmetic preparations. With these cosmetic preparations can be promoted according to the formation of disulfide bridges, for example, hair.

The cosmetic preparations may contain, in addition to the previously described heterocyclic compound, suitable solvents as well as additives customary in such formulations. Examples include emulsifiers and coemulsifiers, surfactants, oil, preservatives, perfume oils, cosmetic care and active ingredients such as AHA acids, fruit acids, ceramides, phytantriol, collagen, vitamins and pro-vitamins, such as vitamins A, E and C, retinol, bisabolol , Panthenol, natural and synthetic light stabilizers, natural substances, opacifiers, micropigments such as titanium oxide or zinc oxide, superfatting agents, pearlescent waxes, bodying agents, thickeners, solubilizers, complexing agents, fats, waxes, silicone compounds, hydrotropes, dyes, stabilizers, pH regulators, reflectors, Proteins and protein hydrolysates, protein hydrolysates, salts, gelling agents, bodying agents, silicones, humectants, moisturizers and other common additives. Furthermore, polymers can be contained to adjust the respective desired properties. In order to protect the hair from being impaired by UV radiation, UV light stabilizers may also be present in the cosmetic preparations.

Hair cosmetic preparations comprise, in particular, styling agents and / or conditioners in hair cosmetic preparations, such as hair treatments, hair foams,

Hair gels, hair sprays, hair lotions, hair conditioners, hair shampoos, hair emulsions,

Leveling agents for hairwashing, hair dyeing and bleaching agents, setting lotions or similar products. Depending on the application, the hair cosmetic

Preparations as (Erosol) spray, (Erosol) foam, gel, gel spray, cream, lotion, milk or wax are applied.

Preferably, the agent is a product for the hair selected from hair shampoo and hair products which are rinsed or not rinsed and applied before or after shampooing, coloring, decolorization, perming or wrinkling.

According to the invention therefore also a method for the treatment of hair is provided, which is characterized in that the hair with the cosmetic agent, containing at least one of the previously explained in more detail heterocyclic compound are brought into contact and optionally rinsed with water. The heterocyclic compound is preferably selected from the compounds and classes of compounds detailed above.

Furthermore, as stated, the method can also be used to form disulfide bridges of synthetic substances which have only functional groups bearing SH groups, but are not constructed from amino acids, for example (but for example from an organic polymer).

According to an advantageous further development, the separation of the substance promoting the formation of disulfide bridges is considerably facilitated. According to this idea, a carrier is loaded with the disulfide bridge promoting substance. The carrier may, for example, be a (hydrophilic) resin. The binding of the substance to the carrier has the consequence that the separation of the supported substance, for example. By simple

Filtration is possible. Therefore, it may be advantageous in the above disulfide bond forming process to use caffeine or caffeine-like substances (see above)

Formulas) bound to a carrier to facilitate the separation.

In order to facilitate the binding of the substance to the carrier, functional groups on the substance are helpful. Therefore, derivatives of the substance forming the disulfide bridges can also be used. Examples of suitable caffeine derivatives are:

Theophylline-8-butyric

and

Theophylline-7-acetic acid

Both derivatives promote the formation of disulfide bridges and thus the cyclization of peptides in solution. When these substances are covalently linked to a suitable carrier via their functional group, an immobilized reagent capable of accelerating the closure of disulfide bridges is obtained. After the reaction, the reagent can be removed by simple filtration from the reaction solution. As is clear from these compounds, it is also possible according to the invention, to the remaining positions on the heterocyclic ring and independently of the radicals R ₁ 'to R ₃ ' radicals, as here in the case of 8- (3-Carbo xypropyl) -1 For example, 3-dimethylxanthine, for example, a functional group as R ₅ 'for coupling to the carrier, attach.

The invention further relates to the use of the previously described heterocyclic compounds for the formation of disulfide bridges, in particular of intra- or intermolecular disulfide bridges in peptides and proteins.

The substances to be used according to the invention are particularly suitable for the cyclization of peptides, in particular EPO mimetic peptides, by the formation of intramolecular disulfide bridges. Particularly preferred examples are N-methyl-2-pyridone, 2,6-dihydroxy-pyridine hydrochloride, uracil-6-carboxylic acid, 2,4-dihydroxy-6-methylpyrimidine, 2,4-dimethyl-6-hydroxypyrimidine, 2 isopropyl-6-methyl-4-pyrimidinol, 4,6-dihydroxy-2-methylpyrimidine, 4,6-dihydroxypyrimidine, 1, 2-dihydro-3,6-pyridazinedione, 7-hydroxy-5-methyl [1, 2, 4] triazolo [1,5-a] pyrimidine, barbituric acid, alloxan monohydrate and violuric acid, uracil, 1-methyl-uracil, 3-methylxanthine, theobromine, theophylline, caffeine, isocaffeine, xanthine, theophylline-7-acetic acid, theophylline 8-butyric acid, 3-isobutyl-1-methylxanthine, 1, 2,3-benzotriazine-4 (3H) -one, (-) - riboflavin, lumazine, alloxazine, minoxidil (= 6- (1-piperidinyl) -2, 4-pyrimidinediamine-3-oxide) and aminexil (= 2,4-diaminopyrimidine-3-oxide).

These substances can be used according to the invention particularly well for the formation of disulfide bridges. Particularly preferred are:

N-methyl-2-pyridone, barbituric acid, alloxan monohydrate, violuric acid, 4,6-dihydroxy-pyrimidine, uracil-6-carboxylic acid, minoxidil, 3-methylxanthine, theobromine, theophylline, 3-isobutyl-1-methylxanthine, caffeine, Isocaffeine, lumazine, alloxazine.

Such disulfide bridges are formed between SH-containing groups. In particular, natural and non-natural amino acids which have free SH groups are suitable disulfide bridge formers.

Because of their ability to promote the formation of disulfide bridges, the substances of the above formula can be used, for example, for the treatment of SH-containing substances and materials to promote the formation of disulfide bridges. Thus, the substances can be used, for example, for the treatment of hair or fibers (natural and Synthetikfasem). This applies in particular to cysteine-containing fibers. The heterocyclic compounds to be used according to the invention can also be used, for example, in liquid formulations (for example in the form of rinses or shampoos or other agents for treating hair such as, for example, perming agents). Corresponding compositions are therefore also encompassed by the invention.

Due to the catalytic effect of heterocyclic compounds characterized according to the invention on the formation of disulfide bridges, they can also be used, for example, for catalysis in the formation of intermolecular or intramolecular disulfide bridges for the preparation of dynamic combinatorial libraries. They can therefore serve to form disulfide bridges between synthetic or natural or modified natural molecules. Therefore, they can find application in the production of dynamic combinatorial libraries for drug discovery. For dynamic combinatorial libraries, the individual units often crosslinked via disulfide bridges to macromolecules (see Fig. 15). Details of the libraries are described, for example, in "Dynamic combinatorial libraries of macrocyclic disulfides in water" by S. Otto, RLE Furlan and JKM Sanders, J. Amer. Chem. Soc., 2000, 122, 12063-12064 ";"Selection and amplification of hosts from dynamic combinatorial libraries of macrocyclic disulfides. S. Otto, RLE Furlan and JKM Sanders, Science, 2002, 297, 590-593 ";" Drug discovery by dynamic combinatorial libraries. Ramström, Lehn. Nat Rev Drug Discov. 2002 "and WO01 / 64605.

The process according to the invention will be explained below with reference to a few examples. EPO mimetic peptides and oxytocin were chosen as examples of the peptides that can be cyclized by the method according to the invention.

Fig. 1

Shown is the course of the reaction of the cyclization of an EPO-mimetic peptide of the following sequence

GGTYSCHFGKLTWVCKKQGG-Am (BB57)

(0.7 mg / ml) to the corresponding cyclized product in the presence of caffeine (0.3 mg / ml) with air in water at room temperature. As the curve clearly shows, the reaction rate increases in the course of the reaction, suggesting an autocatalytic mechanism of the reaction. The abbreviation Am generally stands for an amidation.

Fig. 2 shows the cyclization of the same peptide as in Fig. 1 (0.7 mg / ml) to its cyclized form in the absence of caffeine. As can be clearly seen, the reaction rate is considerably reduced.

In the case of EPO-mimetic peptides, an optimal concentration of caffeine has been found to be in the range 0.075 to 0.15 mg / ml. Sales are already quantitative after ten hours.

Fig. 3

Depicted is the dependence of the rate of conversion of the EPO-mimetic peptide shown in FIG. 1 on the caffeine concentration. As shown, very good results are achievable in a concentration range of 0.03 mg / ml to 0.3 mg / ml. The optimal values are in the range of 0.06 mg / ml and 0.075 and 0.15 mg / ml, respectively.

Fig. 4 Shown is the dependence of Zyklisierungsgeschwindigkeit on pH. As can be clearly seen, the autocatalytic course of the reaction is not due to a change in the pH, since this effect also occurs in the illustrated buffered solutions (phosphate buffer, pH 6 to 9). However, it has been shown that the yield of cyclized peptide decreases at higher pH values. Furthermore, it has surprisingly been found that the reaction proceeds faster, the lower the pH of the solution. Therefore, a lower pH of below 7 and preferably <= 6.5 is preferred.

Fig. 5

Shown is the influence of the mild oxidizing agent glutathione (oxidized form) on the reaction rate. Here, the reaction of the peptide shown in FIG. 1 (0.7 mg / ml H ₂ O) in the presence of 0.1 mg / ml (0.5 equivalents) of glutathione, oxidized form (GSSG) and caffeine (0.3 mg / ml). Within five to six hours, the reaction was already complete. The reaction of the peptide only with GSSG (in the absence of caffeine) is slow. It has also been shown that undesired by-products are formed (see Fig. 6).

FIG. 6 shows chromatograms which were recorded in each case after one hour of reaction and which confirm the reaction of the EPO-mimetic peptide used with 0.5 equivalents of GSSG.

FIG. 7 shows a tabular overview which compares the method according to the invention with the methods known in the prior art. The tested peptides had the following sequence:

EMP1: Ac-GGTYSCHFGPLTWVCKPQGG-At APG1: Ac-GGTYSCHFGKLTWVCKKQGG-At APG2: Ac-GGTYSCHFGKLT-NaI-VCKKQRG-Am

The in vitro experiments showed a comparable activity of the peptides, which were cycled by the different methods. However, the method according to the invention is distinguished by better yields and purities in relation to the other cyclization methods tested, as clearly demonstrated in FIG. Another advantage of the method according to the invention is that the cyclization reagent used can be separated off in a simple manner by HPLC. Further examples of EPO-mimetic peptides cycled by the method according to the invention are listed below:

Ac-C (tBu) -GGTYSCHFGKLT-Nal1 -VCKKQRG-GGTYSCHFGKLT-NaH -VCKKQRG-Am (APG3) AC-C (MOb) -GGYCFGKLT-NaII-VCKKQRG-GGTYSCHFGKLT-NaII-VCKKQRG-Am (APG4)

Ac-C (tBu) -GGTYSCHFGKLTWVCKKQGG-GGTYSCHFGKLTWVCKKQGG-Am (APG5) (Sama) -GGTYSCHFGKLT-Nai -VCKKQRG-GGYYGFGKLT-NaI -VCKKQRG-Am (APG6)

FIG. 8 Shown is the cyclization of dimeric EPO mimetic peptides. The cyclization of di- or multimeric peptides is preferably carried out in several steps. FIG. 8 shows the synthesis scheme based on a bivalent (dimeric) EPO-mimetic peptide, which is cyclized in 2 steps by formation of two intramolecular disulfide bridges. According to this method, the first disulfide bridge is formed by the method according to the invention. The second intramolecular disulfide bridge was performed using optimized iodine oxidation. For coupling of the peptides to a polymeric carrier, further cysteine residues have also been partially introduced into the molecule. This cysteine was protected by suitable protecting groups tBu or Mob).

The first cyclization using caffeine according to the invention is preferably carried out at pH 6, while the second cyclization was carried out in 80% acetic acid according to the example shown. The yield of the synthesis was typically between 60 and 90%.

Fig. 9

Some of the EPO mimetic peptides are very difficult to cyclize. An example of this is the following peptide:

Ac -GGTYSCHFG-Har-LT-1 -NaI-VCK-Aad-Q-Aad-G-NH ₂

Har = homoarginine

Aad = 2-aminoadipic acid, "homoglutamic acid" NaI: naphthylalanine

In this sequence, it has been found that increasing the concentration of cyclization reagent is advantageous. Thus, the cyclization with 10mg / ml caffeine was successful within 24h. The results are shown in FIG. The yield after about 21 h in solution was already> 90%. 10 to 12

Besides EPO mimetic peptides, a reduced peptide derived from oxytocin was also cyclized with caffeine or the caffeine-like substance (see above formula).

In the course of the reaction, oxytocin, reduced (OyxR), crude product, was dissolved in water (or H ₂ O / ACN / TFA) and allowed to stand in air with various concentrations of caffeine (and possibly GSSG). The reaction batch was measured by HPLC at regular intervals to determine the levels of OxyR and the product oxytocin (oxy).

The tables shown in FIGS. 10 to 12 and the accompanying graphs provide a summary of the results.

It can be seen that the product yield correlates with the peptide concentration. Lower amounts of peptide lead to better results with oxytocin.

The reaction time until complete conversion of OxyR correlates with the concentration of caffeine in the reaction solution. Up to a concentration of 0.5 mg / ml caffeine, the more caffeine the faster the oxidation proceeds. The peptide concentration has only a minor influence on the duration of the reaction.

GSSG does not seem to affect speed or yield.

OxyR, HPLC purified, spontaneously cyclizes "quite" fast, but the rate of reaction can be significantly shortened with caffeine again Small amounts of ACN / TFA have little effect on the yield, the reaction time is slightly prolonged.

FIGS. 13 and 14

Shown are the results of the cyclization of the peptide BB57 with the substance minoxidil:

To this was dissolved 0.7 mg of BB57 and 0.3 mg of minoxidil (6- (1-piperidinyl) -2,4-pyrimidinediamine 3-oxide, Minox) in 1 ml of distilled water and allowed to stand in air. Of the Sales of BB57 to oxidized BB57C was monitored by HPLC (UV detection at 216nm). The results are shown in FIG.

As shown in FIG. 13, the oxidation in the presence of minoxidil is completed after about 29 h (in a comparison measurement with caffeine it is about 24 h). Minoxidil, as a further representative of the heterocyclic compounds according to the invention, therefore likewise has a positive effect on the formation of disulfide bridges. The yield in solution of the minoxidil-catalyzed reaction is over 95%. The fact that the reaction is a catalytically occurring reaction is shown by the fact that the concentration of minoxidil in the reaction does not decrease practically (decrease <2% at 4.4 eq minoxidil based on BB57, see FIG. 14).

Fig. 15

Shown are possible linkage strategies via disulfide bridges for generating crosslinked macromolecules. Such devices are often used in dynamic combinatorial libraries.

Fig. 16

Furthermore, experiments were carried out to demonstrate that hair, which were previously reduced in terms of a permanent wave treatment, with a combination of the substance according to the invention (here caffeine) and an additional

Oxidizing agent (here oxidized glutathione-GSSG) in the presence of air accelerated oxidatively close. Thus, the method according to the invention can be used advantageously also in the cosmetic field and in particular in the hairdressing sector for the treatment of hair.

The experiment was carried out as follows:

Each 5-6 mg of hair is mixed with 400 μl of a 10% solution of an ammonium thioglycolate-containing "well lotion" (product "Poly Lock - strong perm", Schwarzkopf & Henkel,

Germany) 0.5 h at room temperature. The solution is removed and the hair is then washed 6 times with 400 μl of H ₂ O each time. One each of them treated that way

Hair samples are a) in H ₂ O, or an aqueous solution of b) 10 mg / ml caffeine, c)

5mg / ml GSSG in H ₂ O and d) 10mg / ml caffeine and 5mg / ml GSSG at room temperature for 3 days.

To determine the free, remaining thiol groups, the hair after removal of the reaction solution with Ellman's reagent (5,5'-dithiobis (2-nitrobenzoic acid), DTNB) is reacted. As additional reference samples are untreated hair and reduced hair, which was otherwise subjected to no further treatment. The hair samples are each with 200μl one Add 10 mM phosphate buffer, pH 8.0 and 1 mM EDTA, and 300 μl of a 1 mM DTNB solution in the same EDTA-containing buffer. The solution is measured after a few minutes in a UV-Vis spectrometer.

The UV-Vis spectra of the samples show that the reduction of hair was successful by treatment with the ammonium thiol glycol containing "well lotion" (see Figure 29, curves e and f showing untreated and once reduced hairs) can be seen from the fact that the band is greatest at 412 nm - a measure of the number of free thiol groups - a treatment in the presence of air with a) H ₂ O, b), with a caffeinated solution, c) the GSSG-containing However, the solution leads only to a partial oxidation of the thiol groups, but it can already be seen here that the caffeine has a positive influence on the closure of the disulfide bridges, but the combination d) of caffeine and the oxidant GSSG led to an almost quantitative oxidation of the thiol groups This experiment therefore proves that the method according to the invention for closing disulfide bridges can also be used successfully on hair.

FIGS. 17 to 39

Shown are the results of the cyclization of the reference peptide BB57 with various substances that can promote the formation of disulfide bridges according to the invention.

The experiments were carried out as previously stated (see above). The substance tested and the quantities tested are indicated.

Claims

claims:

1. A process for the formation of disulfide bridges, characterized in that the reaction is carried out in a liquid medium containing at least one compound which promotes the formation of disulfide bridges, said

Connection selected from the following group:

(a) a compound having in its structure a saturated or unsaturated six-membered heterocycle having at least one nitrogen atom, said heterocycle being attached to the

Nitrogen atom adjacent carbon atom has at least one hydroxy group or oxo group (= 0) according to the invention, wherein in the case of the presence of a hydroxy group, the heterocycle is unsaturated;

(b) a compound of the following general formula

in which the substituent A is hydrogen, an optionally substituted alkyl radical, an optionally substituted aryl radical or a saturated or unsaturated heterocyclyl having 3 to 10 ring members and 1 to 3 heteroatoms, such as nitrogen, oxygen and / or sulfur, wherein the heterocyclyl unsubstituted or monosubstituted or polysubstituted by halogen, alkyl having 1 to 4 carbon atoms, cyano, nitro, cycloalkyl having 3 to 6 carbon atoms, hydroxy, alkoxy having 1 to 4 carbon atoms and / or mercapto.

2. The method according to claim 1, characterized in that the compound according to alternative (a) has the following basic structure

V, W, X, Y and Z are either carbon atoms or nitrogen atoms, the heterocycle having in total not more than three nitrogen atoms;

- R2 and R3, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is hydrogen or alkyl, an electron-withdrawing substituent, a functional group in particular a hydroxy group, an oxo group (= O) according to the invention, -CONH ₂ or an oxime (= N-OH) or R ₂ together with R _{3 represents} a five- or six-membered ring which may likewise have heteroatoms and optionally carries further substituents, or R2 and / or R3 are missing;

- R4 and R5, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an electron-withdrawing one

Substituents, a functional group such as in particular a hydroxy group, an oxo group according to the invention (= 0), a carboxy group, -CONH ₂ or an oxime (= N-OH) or R4 together with R5 represents a five- or six-membered ring, which is also heteroatoms and optionally carries further substituents, R5 together with R6 represents a five- or six-membered ring, which may also have heteroatoms and optionally carries further substituents or R4 and / or R5 is missing;

- R6 is hydrogen or an optionally substituted alkyl or

Aryl radical, or R6 together with R5 is a five- or six-membered Ring, which may also have heteroatoms and optionally carries further substituents, or R6 is missing.

3. The method according to claim 2, characterized in that the compound contains the following substructure

wherein the heterocycle is saturated or unsaturated depending on the choice of the substituents R 2, R 3, R 4 and R 6 and correspondingly one or more

May have double bonds; in which

- R2 and R3, each independently of one another represent hydrogen, an optionally substituted alkyl or aryl radical, an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is the same

Are hydrogen or alkyl, an electron-withdrawing substituent, a functional group such as in particular a hydroxy group, an oxo group (= O) according to the invention, -CONH ₂ or an oxime (= N-OH) or R ₂ together with R _{3 represents} a five- or six-membered ring, which may also have heteroatoms and optionally bears further substituents, or R2 and / or R3 are missing;

- R4 is hydrogen or an optionally substituted alkyl or aryl radical or R4 is absent;

4. The method according to claim 3, characterized in that the compound represents a uracil derivative of the following general formula:

5. The method according to claim 3, characterized in that it is purine derivatives in the compound whose skeleton corresponds to the following general formula

in which the five-membered ring is unsaturated and correspondingly has double bonds and in which the substituents

R4 and R6, each independently, are hydrogen, an optionally substituted alkyl group or an optionally substituted aryl group; preferably hydrogen, an optionally substituted Ci to Ci _o alkyl or an optionally substituted C ₆ - or C _O-aryl radical; particularly preferably hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical or an optionally substituted C ₆ -aryl radical; in particular hydrogen or an optionally substituted C ₁ to C ₃ alkyl radical;

- R7, R8 and R9, each independently of one another, represent hydrogen, an optionally substituted alkyl radical, an optionally substituted aryl radical or an optionally substituted radical - (CH ₂ ) _n COOX where n is 0 to 10 and X is hydrogen or alkyl or a functional Stand group; preferably hydrogen, an optionally substituted C ₁ - to C ₁₀ -alkyl radical, an optionally substituted C ₆ - or C ₁₀ -aryl radical or an optionally substituted radical - {CH ₂ ) _n -COOX with n = 1 to 10 and X is hydrogen or C ₁ to C ₈ alkyl; particularly preferably hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical, an optionally substituted C ₆ -aryl radical or an optionally substituted radical - (CH ₂ ) _n -COOX with n = 1 to 6 and X is the same Hydrogen or C ₁ - to C ₆ -alkyl; in particular hydrogen, an optionally substituted C ₁ to C ₃ -alkyl radical or an optionally substituted radical - (CH ₂ ) _n -COOX with n = 1 to 4 and X is hydrogen or C ₁ - to C ₃ -alkyl.

6. The method according to any one of claims 1 to 3, characterized in that R2 and R3 together form a six-membered ring which optionally has at least one heteroatom.

7. The method according to claim 6, characterized in that the compound has the following basic structure:

the rings being unsaturated depending on the choice of the substituents and correspondingly being able to carry one or more double bonds,

R1 represents either a hydroxy group or an oxo group of the invention (= 0);

R4 and R6, each independently of the other, are or are absent for hydrogen, an optionally substituted alkyl radical or an optionally substituted aryl radical; are preferably hydrogen, an optionally substituted branched or unbranched C ₁ - to C ₁₀ -alkyl radical or an optionally substituted C ₆ - or C ₁₀ - aryl radical; particularly preferably hydrogen, an optionally substituted C ₁ - to C ₆ -alkyl radical or an optionally substituted C ₆ -aryl radical; in particular hydrogen or a substituted or unsubstituted C ₁ to C ₃ alkyl radical;

R5 is hydrogen, an optionally substituted alkyl or aryl radical, an electron-withdrawing substituent, a functional group such as in particular a hydroxy group, an oxo group (= 0) according to the invention, a carboxy group, -CONH ₂ or an oxime (= N-OH) or R5 is missing; R10 and R13, each independently of one another, are hydrogen, an optionally substituted alkyl radical or an optionally substituted aryl radical, or R10 and / or R13 are absent; preferably hydrogen, an optionally substituted branched or unbranched d- to Ci _O alkyl or an optionally substituted C ₆ - or Cio-aryl; particularly preferably hydrogen, an optionally substituted (C ₁ to C ₆ -alkyl radical or an optionally substituted C ₆ -aryl radical, in particular hydrogen or a C ₁ to C ₆ substituted by at least one hydroxyl group,

Alkyl radical;

R11 and R12 each independently represent hydrogen, an optionally substituted alkyl or aryl group, an electron-withdrawing substituent, a functional group such as

Hydroxy group, an oxo group according to the invention (= 0), a carboxy group, -CONH ₂ or an oxime (= N-OH) or R11 and R12 together form a five- or six-membered ring, which may optionally carry further heteroatoms and substituents.

8. The method according to one or more of claims 1 to 7, characterized in that the compound according to alternative (b) has a substituent A, the

- is hydrogen, an optionally substituted C 1 to C ₁₀ alkyl radical, an optionally substituted C ₆ or C ₁₀ aryl radical or a saturated or unsaturated heterocyclyl having 3 to 10 ring members and 1 heteroatom, such as nitrogen, oxygen and / or sulfur where the heterocyclyl is unsubstituted or monosubstituted or polysubstituted by halogen, alkyl having 1 to 4 carbon atoms, cyano, nitro,

Cycloalkyl of 3 to 6 carbon atoms, hydroxy, alkoxy of 1 to 4 carbon atoms and / or mercapto;

preferably hydrogen, an optionally substituted C ₁ - to Ce-alkyl radical, an optionally substituted C ₆ -aryl radical or saturated heterocyclyl having 5 or 6 ring members and 1 heteroatom, such as

Nitrogen, oxygen and / or sulfur, wherein the heterocyclyl is unsubstituted or monosubstituted or polysubstituted by halogen,

Alkyl of 1 to 4 carbon atoms, cyano, nitro, cycloalkyl of 3 to 6 carbon atoms, hydroxy, alkoxy of 1 to 4 carbon atoms and / or mercapto; in particular hydrogen, an optionally substituted C ₁ to C ₃ alkyl radical or saturated heterocyclyl having 5 or 6 ring members and 1 heteroatom, such as nitrogen, oxygen and / or sulfur, wherein the heterocyclyl is unsubstituted;

stands.

9. The method according to one or more of the preceding claims, character- ized in that the compound to promote the training of

Disulfide bridges having the following structure

wherein Rv, R ₂ ', R ₃ ' are the same or different and at least one of R _r , R ₂ ', R ₃ ' is an alkyl group.

10. The method according to one or more of claims 1 to 9, characterized in that Rv, R ₂ ', R ₃ ' are the same or different and are either hydrogen or a Ci to C ₅ alkyl group, in particular a Ci to C ₃ alkyl group, most preferably represent a methyl group.

11. The method according to one or more of claims 1 to 10, characterized in that the compound is selected from the group consisting of N-methyl-2-pyridone, 2,6-dihydroxy-pyridine hydrochloride, uracil-6-carboxylic acid, 2,4-dihydroxy-6-methylpyrimidine, 2,4-dimethyl-6-hydroxypyrimidine,

2-isopropyl-6-methyl-4-pyrimidinol, 4,6-dihydroxy-2-methylpyrimidine, 4,6-dihydroxypyrimidine, 1, 2-dihydro-3,6-pyridazinedione, 7-hydroxy-5-methyl [1, 2,4] triazolo [1,5-a] pyrimidine, barbituric acid, alloxane monohydrate and violuric acid, uracil, 1-methyl-uracil, 3-methylxanthine, theobromine, theophylline, caffeine, isocaffeine, xanthine, theophylline-7-acetic acid, theophylline-8

Butyric acid, 3-isobutyl-1-methylxanthine, 1, 2,3-benzotriazine-4 (3H) -one, (-) - riboflavin, lumazine, alloxazine, minoxidil and aminexil.

12. The method according to one or more of claims 1 to 11, characterized in that at least one intramolecular disulfide bridge in amino acid-containing substances, in particular in peptides and proteins, is formed, wherein the reaction is carried out in an aqueous medium.

13. The method according to one or more of claims 1 to 12, characterized in that an intramolecular disulfide bridges is formed between two amino acids having an SH group, preferably between two cysteine residues.

14. The method according to one or more of claims 1 to 13, characterized in that the reaction mixture, an oxidizing agent, preferably glutathione in oxidized form, is added.

15. The method according to one or more of claims 1 to 14, characterized in that the peptides have a length between 5 and 100, 5 and 50 amino acids, preferably between 10 and 40, particularly preferably between 15 and 25 amino acids.

16. The method according to one or more of claims 1 to 15, characterized in that the substance is bound to a carrier.

17. The method according to claim 16, characterized in that the substance is bound via functional groups to the carrier.

18. Use of a heterocyclic compound according to any one of claims 1 to 12 for the formation of disulfide bridges.

19. Use according to claim 18, characterized in that the heterocyclic compound is used for the cyclization of peptides and / or proteins, wherein the peptides preferably have a length between 5 and 250, 5 and 100, 5 and 50, preferably 10 to 40, especially preferably between 15 and 25 amino acids.

20. Use according to claim 18 or 19, characterized in that the substance is used for the cyclization of cytokine-mimetic peptides, in particular EPO-mimetic or TPO-mimetic peptides.

21. Use according to one or more of claims 18 to 20 for forming an intramolecular disulfide bridge between at least two SH-groups bearing amino acids.

22. Use according to one or more of claims 18 to 21 for the treatment of SH-group-carrying substances and products for the formation of disulfide bridges.

23. Use according to claim 22 for the treatment of hair and fibers, in particular cysteine-containing fibers.

24. A composition comprising a heterocyclic compound according to one or more of claims 1 to 11 for the formation and / or promotion of disulfide bridges.

25. Use of the composition according to claim 24 for the treatment of hair and / or fibers.

26. Use of a heterocyclic compound according to any one of claims 1 to 11 for catalysis in the formation of inter- or intramolecular disulfide bridges for the production of dynamic combinatorial libraries.

27. Cosmetic agent for promoting the formation of disulfide bridges, characterized in that it contains a heterocyclic compound having at least two nitrogen atoms, according to at least one of

Claims 1 to 12.

28. A method for treating hair, characterized in that the hair with the cosmetic composition according to claim 28 are brought into contact and optionally rinsed with water.