WO2004014941A1

WO2004014941A1 - Method for selective peptide labeling

Info

Publication number: WO2004014941A1
Application number: PCT/DE2003/002534
Authority: WO
Inventors: Norman Koglin; Manja Lang; Annette Beck-Sickinger
Original assignee: Universität Leipzig
Priority date: 2002-07-23
Filing date: 2003-07-23
Publication date: 2004-02-19
Also published as: WO2004014941A8; DE10334499A1; AU2003258472A1; AU2003258472A8

Abstract

The invention relates to a method for selective peptide labeling, whereby the step of selective, radioactive labeling of individual amino groups of peptides in a solution is particularly made possible. Peptides from the microgram (µg) scale onwards can be selectively and radioactively labelled regardless of their sequence. An amino acid building block is inserted during peptide synthesis in at least one position that is to be subsequently labelled, said building block carrying an amino group that is protected by a group functionally different from the protective groups on the amino groups that are not be labelled. Following peptide synthesis, the protective group in the position to be labelled is selectively removed and the peptide is separated from the carrier material. A label is then introduced in the so obtained deprotected peptide by means of amino reactive substances at the position to be labelled. The protective groups on the amino groups that are not to be labelled are removed in a simple manner after labeling, preferably by means of ultraviolet light, and the resulting selectively labelled peptide is purified using a conventional method.

Description

Process for the selective labeling of peptides

The invention relates to a method for the selective labeling of peptides for use in biotechnological research, the pharmaceutical industry and medical diagnostics. Especially in the post-genome age, new and selectively labeled biomolecules are always required to carry out bioactivity studies. So one needs for the investigation of the interaction of peptides z. B. with peptide receptors in binding studies and for structure-activity relationships at certain positions radio-labeled and thus easily detectable peptides. However, this label must not restrict biological activity. They have radioactively labeled peptides compared to e.g. B. fluorescence-labeled peptides have the advantage that they are highly sensitive detectable and change the biological and physicochemical properties of the biomolecules less. ^[1 ' ² - ¹

According to the state of the art, peptides are usually produced by chemical peptide synthesis. A peptide is built up sequentially on a solid support material (resin). The most commonly used isotopes in biochemistry as radioactive markers are ¹²⁵ iodine ( ^I25 I), ³⁵ sulfur ( ³⁵ S) and tritium ( ³ H). The methods for incorporating these radioactive markers into the peptide differentiate between direct and indirect labeling methods.

A direct marking is done e.g. B. by the use of radioactively labeled amino acids (z. B. ^{3: ι} S-methionine) for peptide synthesis. The disadvantage here is that these labeled amino acids are very expensive and are also only available to a limited extent with protective groups suitable for peptide synthesis. It is also problematic that the use of radioactively labeled amino acids in the peptide synthesis has the consequence that the entire synthesis and a z. T. elaborate processing must take place in a radioactive laboratory.

Alternatively, direct labeling following peptide synthesis by isotope exchange with a radioactive isotope, e.g. B. with tritium ( ³ H). However, this requires reaction conditions which are not very suitable for peptides (e.g. high temperature rature) and reagents that may only be used in specially equipped laboratories and under strict safety requirements.

Indirect labeling of peptides is preferably carried out by derivatization of primary amino groups (ie the N-terminus or α-amino group of lysine residues) by means of the Bolton-Hunter reaction. Radioactive acyl residues activated by N-hydroxysuccinimide (NHS) esters are transferred to amino groups. ^[3] Such NHS esters of radioactive acyl residues are commercially available, such as. B. the tritium-labeled [2,3- ³ H] propionyl NHS ester. The reaction scheme for labeling a lysine residue with this NHS ester is shown in Formula 1.

(Formula 1) Indirect labeling of peptides can either be carried out a.) With a peptide which is still attached to the support material after synthesis and is side-chain protected except for the site to be labeled, or b.) With a free peptide in solution.

The advantage of radioactive labeling of a peptide bound to the carrier material (resin) is the possibility of selectively deprotecting and derivatizing amino acid side chains. This advantage is, however, after the synthesis by z. T. elaborate work steps lost under radioactive conditions. The disadvantage here is that resin-bound peptides can only be used with approaches from the milligram range. Therefore, radioactive labeling on the resin requires considerable amounts of radioactivity and is very expensive.

Since a cleaning step of the peptides before the radioactive labeling on the resin is not possible, when carrying out these labor-intensive and device-intensive reprocessing steps (e.g. in preparative HPLC), high losses of the desired peptide and thus also of the radioactivity used can sometimes occur. Especially at peptide sequences that are difficult to synthesize and thus more heavily contaminated raw peptides are reprocessed, and a large part of the radioactivity used is lost again due to likewise marked incorrect sequences. The use of large amounts of radioactivity is also a high cost, requires special permits, also poses a higher health risk and can endanger the environment through larger amounts of waste. They also require a high level of equipment, especially for the processing and analysis of the radioactively labeled peptides.

However, a few micrograms of a high-affinity radio-labeled peptide are usually sufficient for numerous purposes. Furthermore, different peptides marked at defined positions are often required in screening studies, particularly in new receptor-ligand interaction studies. In most cases, it is therefore advisable to initially display and use only small amounts of a radioactive biomolecule for testing.

With indirect labeling in solution, the peptide is radioactively labeled according to the prior art after the peptide synthesis after the removal of all protective groups.

This also has advantages and disadvantages. The advantage is that even very small quantities (μg range) can be handled. However, it is impossible to selectively label individual amino acids as soon as the peptide contains several reactive groups of the same type (e.g. several amino groups). This is often the case with longer peptides.

A selective marking is u. a. necessary if individual reactive groups are not allowed to be modified for the full biological activity of the peptide.

Several free amino groups are present in the peptide sequence as soon as the peptide contains one or more lysine residues (Lys or K) in addition to the mostly free N-terminus.

A chemical distinction between two lysines is impossible / Attempting a chemical distinction between free N-terminus and α-amino groups of lysines results in inhomogeneously labeled peptides. ^[4] When labeling peptides in solution, selective labeling of individual Lys residues is therefore impossible. The separation of unselectively labeled peptides is associated with high losses and effort and is often only possible with dubious quality.

The object of the present invention is to provide a method for the selective labeling of peptides. With this method, in particular, the step of selective radioactive labeling of the peptides in solution should be possible. In addition to the amino group to be labeled, the peptides should also be able to have further amino groups. In particular, it should be possible with the invention to selectively radioactively label peptides regardless of their sequence from the microgram (μg) scale.

According to the invention, the object is achieved by a method for the selective labeling of amino groups in peptides, in which, in a first step, the peptide is produced in a chemical solid-phase synthesis in a manner known per se, wherein

1. in peptide synthesis, at the position to be marked in each case an amino acid building block is installed which bears an amino group which is protected by a group which differs functionally from the protective groups on the amino groups which are not to be marked;

2. after the peptide synthesis, the protective group at the position to be marked is selectively removed and the peptide is detached from the support material,

3. a radioactive label or a label which can be detected by other methods is introduced into the selectively deprotected peptide thus obtained in solution via amino-reactive substances at the position to be labeled, and

4. the protective groups on the amino groups not to be labeled are then split off and the selectively labeled peptide obtained in this way is purified in a known manner.

The method according to the invention can advantageously be handled with just a few μg peptide and is not bound to method-related minimum quantities. The method described below for labeling peptides with radioactive isotopes can also be used to introduce other markers that can be detected using other methods.

With the method according to the invention, homogeneous labeled peptides are obtained in a quality that meets the high requirements for quantitative biological studies. As a result of this method, which is already compatible on a micro scale, the amount of radioactivity to be used for radioactive labeling can be controlled in accordance with the desired amount of radioligand. Another advantage of this method is the reduced number of labor-intensive radioactive work steps and the fact that selective marking can be carried out with less equipment and analytical effort. This method can therefore be cheaper and easier to carry out than previous methods.

The amino group to be labeled selectively can advantageously be chosen freely, i. H. it is either an amino group in the side chain of an amino acid building block (e.g. an ε group of a lysine) or the N-terminus of the peptide built up. Selective labeling on several amino groups is also possible.

Peptides in the sense of this invention are understood to mean organic compounds produced by chemical solid-phase peptide synthesis, which consist of two to approximately one hundred α-, β-, γ-amino acids. These are connected to one another in any order, by means of amide or peptide bonds. In particular embodiments of the peptide synthesis, an amino group (NH) is introduced as the C-terminal amide or a substituted amino group (NHR, R = alkyl) or an alcohol residue at the C-terminus of the peptide. In further embodiments, the peptide is acylated at the N-terminus or glycosylated, phosphorylated or otherwise derivatized at the N- or C-terminus or on side chains.

For peptide synthesis, starting from a free amino acid bound to a solid support (resin) with a terminal free α-amino group (N-terminus) sequentially built a peptide in which one amino acid building block with the C-terminus is added to the free N-terminus of the growing peptide chain per synthesis step. The α-amino group of the added amino acid building block then forms the new N-terminus to which the next amino acid building block is added in the subsequent synthesis step.

So that only the free N-terminus of the amino acid bound to the carrier reacts with the next amino acid building block in the peptide synthesis, amino acid building blocks are used for the synthesis in which, apart from the C-terminus, all chemically reactive groups of the amino acids, such as the α-amino group and other functional ones Groups in the side chains such as amino groups (lysine), or z. B. free hydroxyl groups (tyrosine, serine) are provided with appropriate protective groups.

The protecting group on the α-amino group of the added amino acid building block is selectively removed during the synthesis in order to provide a new free N-terminus for the next synthesis step. The remaining protective groups remain bound and are only removed at the end of the synthesis.

Nowadays peptide synthesis follows either the Boc / Bzl strategy or the Fmoc / ^l Bu strategy. Depending on the synthesis strategy, different protective groups are used.

The Boc / Bzl strategy is characterized by graded acid instability. The α-amino groups are protected by acid-labile tert-butoxycarbonyl groups (Boc groups). During the synthesis, in order to generate a new free N-terminus, the Boc group on the α-amino group is usually cleaved selectively by treatment with trifluoroacetic acid (TFA). The remaining reactive groups of the amino acid side chains, such as. B. hydroxyl groups (Ser, Thr) and ε-amino groups (Lys) are protected by benzyl (Bzl) groups or benzyloxycarbonyl (Z) - groups that z. B. can only be removed by treatment with the stronger hydrofluoric acid (HF). In the orthogonal Fmoc / 'Bu strategy, the α-amino groups are protected by the base-labile 9-fluoro-enylmethoxycarbonyl group (Fmoc group) and the remaining reactive groups of the amino acids are generally protected by acid-labile groups. In this strategy, hydroxyl groups are mostly protected by tertiary butyl groups ( ^{ Bu groups) and the ε-amino groups of lysine by Boc groups. In order to generate a new free N-terminus, the Fmoc group on the α-amino group is treated during the synthesis by treatment with a weak base, e.g. B. 20% piperidine in dimethylformamide (DMF) removed. The remaining protective groups are only removed at the end of the synthesis (e.g. by TFA).

For the peptide synthesis, the orthogonal Fmoc / tert-butyl protective group strategy is preferred. ^]

According to the invention, during the peptide synthesis, an amino acid building block is inserted at at least one position to be marked later, which carries an amino group which is protected by a group which differs functionally from the protective groups on the amino groups which are not to be marked. In addition, the protective group must differ functionally from the protective groups on the amino groups that form the peptide bond.

This functional difference makes it possible to selectively remove the protective group at the position to be marked in a further step of the method.

This functional difference is based on different chemical properties, such as resistance to certain chemical substances.

In the method according to the invention, the amino group to be labeled later is initially protected during the synthesis by a group which can be split off by a method in which the amino protective groups on the amino groups which are not to be labeled are retained. In the method according to the invention, all amino groups which are not to be labeled are preferably protected by photolabile protective groups (for example Nvoc), and only the amino group at the point to be labeled is protected with a chemically labile protective group (SG) such as. B. a benzyloxycarbonyl (Z group in the case of the Boc / Bzl strategy) or a Boc group (in the case of the Fmoc / 'Bu strategy).

In one embodiment of the method, this is achieved in that, in the peptide synthesis, photolabile protective groups, for. B. nitroveratryloxycarbonyl (Nvoc). This is achieved by installing appropriately protected amino acid components, e.g. B. Lysine with Nvoc-protected side chain.

Lysines that are protected on the side chain with Nvoc (N ^ε -Nvoc-lysines) are e.g. B. after reacting N ^α -protected lysines (eg N ^α -Boc-Lys or N -Fmoc-Lys) with Nvoc chloride ^00] .

If the N-terminal amino group is to be selectively labeled, the N-terminal amino group, which is still protected after the last coupling step of the synthesis, is deprotected and the peptide is cleaved from the support material, while all other amino groups not to be labeled in the side chains are removed with one photolabile protecting group are derivatized.

If the N-terminal amino group is not to be selectively labeled, an N ^α -photolabile-protected amino acid is preferably used in the last coupling step of the peptide synthesis. This N ^α -photolably protected amino acid is synthesized analogously to the N ^ε -photolably protected lysine (Vossmeyer: Journal of applied physics 1998). Alternatively, the N-terminal amino group is deprotected after the peptide synthesis and is protected in a subsequent synthesis step by reacting the free N-terminal amino group with the acid chloride of the photolabile protective group (e.g. Nvoc chloride). The photolabile protective groups used in the process according to the invention are preferably groups of the ort / zo-nitrobenzyl type, particularly preferably nitroveratryloxycarbonyl (Nvoc). Its acid chloride (Nvoc chloride) has the following formula (Formula 2):

(Formula 2)

Such photolabile protective groups can be easily removed again by UV radiation at a wavelength of 200-600 nm. ¹ - ^{~ &} compounds of the ortho-nitrobenzyl type undergo photoenolization under the influence of light and there is a fragmentation to ortho-nitrosobenzaldehydes. The lamp strength can vary within the range of 10 W -1000 W. The cleavage is preferably carried out at temperatures between 0 ° C. and 30 ° C. and within 1 minute to 5 hours.

The chemically labile protective group on the amino group to be labeled is selectively removed after the synthesis by hydrofluoric acid (HF) (in the case of the Boc / Bzl strategy) or trifluoroacetic acid (TFA) (in the case of the Fmoc Bu strategy).

By splitting off this chemically labile group by hydrofluoric acid (HF) (in the case of the Boc / Bzl strategy) or trifluoroacetic acid (TFA) (in the case of the Fmoc Bu strategy), the peptide is simultaneously detached from the solid phase support and further protective groups are removed.

The photolabile protective groups on the remaining amino groups that are not to be labeled are not removed in the process. The peptide in solution now only contains a free amino group at the position to be labeled, which is unprotected and can be labeled selectively. This selective labeling takes place, as explained in more detail later, with a radioactive amino-reactive reagent, preferably an N-hydroxysuccinimide (NHS) ester.

The photolabile protective groups on the remaining amino groups are removed by UV light after the labeling. This is shown schematically in Fig. 3_.

However, the direct introduction of amino acid building blocks with photolabile protective groups during peptide synthesis has the disadvantage that the entire synthesis must take place in the dark. Another disadvantage is that amino acids protected with photolabile protecting groups are not yet commercially available.

In a particularly preferred embodiment of the invention, peptide synthesis. Therefore, the photolabile groups are not introduced directly, but previously groups (e.g. Dde) that differ in their reactivity to a chemical agent, such as. B. hydrazine, distinguish from the remaining protective groups (SG). This variant is shown schematically in FIG. 1.

This has the advantage that the chemical peptide synthesis can be carried out with commercially available materials and does not have to take place in the absence of light.

In this embodiment, all amino groups which are not to be labeled with a hydrazine-labile protective group, particularly preferably with 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) or 1, are preferred during the peptide synthesis - (4, 4-dimethyl-2,6-dioxocyclohex-l-ylidene) -3-methylbutyl- (ivDde-) (Novabiochem, Laufelfingen, Switzerland).

For this purpose, in the peptide synthesis for the amino acids that contain amino groups in their side chains, such as. B. Lysine, which should not be labeled, used appropriately protected amino acid building blocks. In these amino acid building blocks, such as. B. a on the ε-amino group protected with Dde lysine (N ^α -Fmoc-N ^ε -Dde-lysine) is Protected amino group, which should not be labeled, with a hydraziήlable Dde protective group (e.g. Dde).

Such amino acids, such as. B. N ^α -Fmoc-N ^ε -Dde-lysine are commercially available (Novabiochem, Laufelfingen, Switzerland).

The amino group to be labeled later, an amino group in a side chain, e.g. B. an ε-amino group of the lysine or the N-terminus, is provided during the synthesis in the case of the Fmoc / 'Bu strategy with an acid-labile, but stable in hydrazine protective group (z. B. Boc). In the case of the Boc / Bzl strategy, the amino group is provided with a base-labile protective group (e.g. Fmoc).

If an amino group in a side chain is to be selectively labeled, in the case of peptide synthesis, in the case of the Fmoc / ^l Bu strategy, an amino acid component which contains an amino group protected with an acid-labile (hydrazine-stable) protective group in the side chain is incorporated in the site to be labeled, z. B. a lysine whose ε-amino group (N ^ε ) is Boc-protected (e.g. N ^α -Fmoc-N ^ε -Boc-lysine).

In the case of the Boc / Bzl strategy, the amino acid component to be selectively labeled accordingly contains an amino group protected with a base-labile protective group (for example Fmoc).

If the N-terminus is to be marked selectively, an amino acid building block is used in the synthesis in the last coupling step in the Fmoc Bu strategy, the α-amino group (N ^α ) of which contains an acid-labile (hydrazine-stable) protective group (e.g. . Boc) is protected. This variant is shown schematically in FIG. 2. In the Boc / Bzl strategy, an amino acid module is used in the last coupling step, the α-amino group of which is protected with a base-labile protective group (e.g. Fmoc).

In the event that the N-terminus is not to be labeled, the protective group which it carries during the last coupling step is removed after the synthesis. removed. In the case of the Fmoc Bu strategy, this is a base-labile protective group (e.g. Fmoc), which is characterized by a weak base, such as. B. 20% piperidine is removed. In the Boc / Bzl strategy, the protective group at the N-terminus which should not be labeled is an acid-labile protective group (e.g. Boc), which is protected by an acid, such as. B. TFA is removed.

The hyrazine-labile protective groups, such as. B. Dde, are directly after the synthesis on the support material with hydrazine, for. B removed with 2% hydrazine in DMF ^[9] .

In an alternative embodiment of the invention, in the Fmoc / tBu strategy, instead of the hydrazine-labile protective groups, protective groups which can be removed under weakly acidic conditions, such as preferably 4-methyltrityl- (Mtt) or 4-methoxytrityl (Mmt), are used.

These protective groups are selectively split off with a dilute acid within 4 times 2 minutes instead of with hydrazine, e.g. B. with 1% TFA in dichloromethane (DCM). Preferably, the dilute acid, a scavenger, preferably 1% to 5% TIS (triisopropylsilane) is added.

Under these weakly acidic conditions, the protective group on the amino acid to be labeled, preferably a Boc group, which can only be removed under strongly acidic conditions (> 50% TFA), is not removed.

The selective cleavage of the protective groups which can be eliminated with hydrazine or under weakly acidic conditions means that all amino groups which are not to be labeled are now freely available. Only the amino group to be labeled is protected. The peptide is still bound to the support material.

All free amino groups are now simultaneously with a photolabile protective group, preferably of the ortΛo-nitrobenzyl type, such as. B. Nvoc provided.

For this purpose, an activated derivative of the photolabile protecting group (e.g. Nvoc chloride), N-hydroxybenzotriazole (HOBt, Novabiochem) and diisopropylethyl min (DIPEA, Fluka, Buchs, Switzerland) and dissolved in N, N'-dimethylformamide (DMF, Biosolve, Valkensward, The Netherlands). The molarity of Nvoc chloride in the reaction solution should be as high as possible (0.1-0.5 M), but the volume should also be sufficient to completely cover the resin. The volume of the respective reaction solution depends on the size of the peptide and is approx. 0.5 ml to 1 ml. The reagents, which are also pre-swollen in DMF, are added to the reagents, which are also dissolved in DMF, and left for at least 3 hours at room temperature or overnight react with shaking. After the reaction has ended, the KAISER test is used to check whether all the free amino groups have reacted. ^u * If this is not the case, the reaction is carried out again.

The introduction of the Nvoc group at all relevant positions on the polymeric support is better and easier to carry out than is the case with the direct use of N ^ε - Nvoc-protected Lys derivatives in the synthesis. ° ⁰ - 'This variant also advantageously does not require the use of special Nvoc-derivatized amino acids which are not commercially available.

After all amino groups that are not to be labeled are protected with a photolabile protective group, the protective group on the amino group to be labeled is removed.

In the case of the Fmoc / 'Bu strategy, the acid-labile protecting group (e.g. Boc) is replaced by an acid, such as. B. TFA removed. In the Boc / Bzl strategy, the base-labile protective group (e.g. Fmoc) is replaced by a weak base, such as. B. 20% piperidine in DMF, removed and the peptide dissolved from the support material.

The peptide now in solution now contains only a free amino group at the position to be labeled, which is unprotected and can be labeled selectively. The photolabile protective groups on the remaining amino groups can be easily removed by UV light after the marking. In the case of the Fmoc Bu strategy, this embodiment of the invention is preferred since N ^ε -Dde-protected lysines, in contrast to N ^ε -Nvoc-protected lysines, are commercially available.

Since the base-labile Fmoc group is not particularly stable, this embodiment is only suitable for building up short peptides using the Boc / Bzl strategy.

In an alternative variant of the invention, therefore, during the synthesis, the relevant amino acid is immediately incorporated and the amino function in question is deprotected from the amino acid. introduced a photolabile protecting group on the amino groups.

This variant is preferred in the Boc / Bzl strategy over the last described embodiment.

In addition, in the peptide synthesis which follows the Boc / Bzl strategy, the amino groups which should not be labeled are protected with base-labile protective groups (e.g. Fmoc). For this purpose, amino acid units provided with base-labile protective groups on their side chains are used for synthesis (e.g. N ^α -Boc-N ^ε -Fmoc-lysine). After the incorporation of each amino acid building block which carries an amino group which is not to be labeled, the base-labile group (e.g. Fmoc) is cleaved off with a weakly basic solution (e.g. 20% piperidine in DMF) and in a subsequent synthesis step, as described above (e.g. by reacting with Nvoc chloride), replaced by a photolabile protective group (e.g. Nvoc). If the N-terminus is not to be protected, its protective group is replaced by a photolabile protective group.

Consequently, at the end of the synthesis, all amino groups which are not to be labeled in the assembled peptide are protected with a photolabile group (such as, for example, Nvoc).

The one in solution after being split off from the polymeric carrier. Peptide now only contains a free amino group at the position to be marked, which is unprotected and can be selectively marked. The photolabile protecting groups on the The remaining amino groups can be easily removed by UV light after the marking.

The peptide, in which the amino group to be labeled is selectively deprotected according to one of the previously described variants of the method according to the invention, is labeled in solution with an amino-reactive substance as follows.

Acylation reagents such as N-hydroxysuccinimide (NHS) esters are preferably used as amino-reactive substances. As described schematically in Formula 1, these NHS esters transfer an acyl group which contains the corresponding marker from the NHS ester to the amino group.

As a marker, the acyl group preferably contains radioactive isotopes, such as. B. ¹ death ( ¹²³ I), ³⁵ sulfur ( ³⁵ S) and tritium ( ³ H).

In further embodiments, other markers are bound to the acyl group, such as the biotin detectable via the affinity for streptavidin / avidin, or immunologically detectable haptens, such as, for. B. digoxygenin or fluorescein.

Dyes which can be detected by fluorescence can also be used as markers, with the use of photolabile protective groups in the process according to the invention followed by the splitting off of the protective group at a wavelength at which the dyes are not bleached.

The labeling reaction can be carried out with just a few micrograms of peptide. Since these small amounts can only be weighed out with difficulty, a stock solution of the peptide purified with preparative HPLC and protected in a photolabile manner was prepared in bidistilled water, the desired amount (e.g. 2 nmol, approx. 10 μg depending on the molecular weight of the peptide) removed from the stock solution and lyophilized.

The labeling reaction can be carried out either in an organic solvent such as DMF or in an aqueous buffer at neutral or slightly basic pH values. leads. For this purpose, the photolabile protected peptide is dissolved in a small amount of 0.1% DIPEA / DMF or in an aqueous and slightly basic buffer. As a buffer z. B. 10 mM phosphate buffer (pH = 7.5) or 10 mM borate buffer (pH = 8.0) can be used, which can optionally be added with low solubility of the peptide acetonitrile (ACN). When labeling at the N-terminus, a slightly acidic buffer (e.g. pH 6.5) can also be used due to the lower pK value of the α-amino group. When choosing the pH value, there is a compromise between the reactivity of the amino group (stronger at higher pH values) and the stability of the labeling reagent in an aqueous medium (lower at higher pH values). ^[13 ^'14]

The peptide is the cheaper component and the nucleophile in the labeling reaction and should be used in a slight excess to increase the yield. Radioactive NHS esters or OSu activatable fluorescent dyes are commercially available (e.g. from Amersham). If the labeling reagent is dissolved in toluene, it is removed in a stream of N ₂ before the reaction. The reaction is preferably carried out in a silanized vessel (for example an Eppendorf tube) in order to keep adsorption of ester and peptide on the vessel wall low. If there is a double excess of peptide (eg 2 nmol), 1 nmol of the labeling substance is used. The peptide dissolved in buffer or DMF is added to the labeling reagent and mixed well. The reaction takes about 1-2 hours at room temperature.

After the labeling, the photolabile protective groups on the amino groups not to be labeled are separated off under the conditions described by means of UV light in an aqueous medium.

In an aqueous environment, it is not necessary to trap unreacted esters beforehand. After the reaction time in the aqueous medium, it can be assumed that the ester has been aminolyzed or is completely hydrolyzed by the side reaction with water and is therefore no longer able to react with the newly released amino groups. After a labeling reaction in DMF, however, a nucleophile such as glycine should be added in order to avoid unselective labeling by unreacted esters after the photolabile protective group has been removed. In order to avoid a greater fragmentation, in particular of Trp-containing peptides, by the UV radiation, the UV exposure should be limited to a maximum of 30 minutes for these peptides. Here too, the photolabile protective group is removed almost quantitatively from the peptide. In order to avoid side reactions, in particular the formation of covalent adducts of the cleaved protective group with the released amino groups, the cleavage is carried out in an acidic environment. For this purpose, the labeling batch is mixed with a 0.1% aqueous TFA / ACN solution. The ACN content can already be adjusted to the subsequent starting conditions of the HPLC gradient.

The peptide is then purified in a known manner. This is preferably done by means of HPLC (High Performance Liquid Chromatography). Since the labeling never runs 100% and, in addition, the Nvoc peptide has been used in excess of radioactive ester, the derivatized peptide must be separated from the non-derivatized peptide. Since the two peptides usually differ only slightly with regard to their elution behavior, depending on the group transferred, a gradient elution with two elution agents was expediently chosen for separation. The gradient may depend on the peptide sequence and the quality of the column used and cannot therefore be generalized for all sequences. The eluents used are: 0.08% TFA in acetonitrile and 0.1% TFA in water.

The following literature gives detailed descriptions of HPLC of peptides: Hancock, W. S .; Sparrow, J.T .: HPLC Analysis of biological compound, Vol 26, 1st edition, Marcel Dekker Inc., New York. 1984

Another object of the invention is the use of peptides in which all amino groups not to be labeled are protected by protective groups, preferably photolabile protective groups, and at least one amino group to be labeled is unprotected for the production of a selectively radioactively labeled peptide. The invention is explained in more detail by the following exemplary embodiments:

Example 1:

Synthesis of Lys (Dde) protected peptides and replacement of the Dde protective group with easily removable photolabile protective groups in solution

The peptide is first synthesized using solid phase peptide synthesis using the orthogonal Fmoc / tert-butyl protecting group strategy on a milligram scale (13.5 μmol). ^f5] For ε-amino groups of Lys side chains, which are still to be protected during the labeling reaction and are to be freely available in later biological studies, a commercially available Lys derivative with 1 - (4,4-dimethyl-2,6- dioxocyclohex-l-ylidene) ethyl (Dde) protected amino function (Novabiochem) used in the side chain. This Dde protective group of the Lys side chain (s) is selectively removed directly on the resin with 2% hydrazine solution in DMF after the synthesis. ^[9] The later to be modified amino group, a certain Lys-side chain or the N-terminus is then still protected with acid labile Boc protecting group. If the N-terminus is marked, an N ^α -Boc-protected amino acid must be used for this in the last coupling step of the peptide synthesis. If, on the other hand, an N ^α -Fmoc-protected amino acid was used in the last coupling step, the N-terminus is already freely available on the resin after synthesis. After removal of the Dde protective group ^) all free amino groups were then provided with the photolabile Nvoc protective group simultaneously.

The photolabile Nvoc group was installed on the resin at the positions that should be free again after the radioactive labeling. Based on the number of free amino functions present, 5 equivalents of 6-nitroveratryloxycarbonyl chloride (Nvoc-Cl, Sigma-Aldrich), 5 equivalents of N-hydroxybenzotriazole (HOBt, Novabiochem) and 10 equivalents of diisopropylethylamine (DIPEA, Flukä) were used. 0.5-1 ml of N, N'-dimethylformamide (DMF, Biosolve) served as solvent. The reagents, which are also dissolved in DMF, are added to the resin which has been swollen in DMF and allowed to react for at least 3 h at room temperature or overnight with shaking. After the reaction is over, check with the KAISER test whether all free amino groups have reacted. ¹ - ¹ ^ If this is not the case, the reaction is carried out again.

After the reaction is complete, the resin is washed thoroughly with DMF, methanol, dichloromethane and diethyl ether and dried in vacuo. The peptide is then cleaved from the resin with 1 ml of trifluoroacetic acid (TFA). The acid-labile side chain protective groups are removed at the same time. A mixture of thioanisole (Fluka) and thiocresol (Fluka) in a ratio of 1: 1 (v / v) is used as the scavenger for the removal of the reactive intermediates that occur. In the case of peptides containing Cys, Met and Trp, a mixture of ethanedithiol (Fluka) and thiocresol (3: 7 (v / v)) is used as the scavenger. The ratio of TFA to scavenger is always 9: 1. The peptides dissolved in TFA are separated from the polymeric support by filtration and precipitated by ice-cooled diethyl ether. The scavenger and protective group fragments are separated by centrifugation and repeated washing with cooled diethyl ether. The peptides are then dried in vacuo, dissolved in a tert-butanol-water mixture and lyophilized. If there are Met residues in the peptide sequence, a subsequent reduction of the z. T. thioether side chains oxidized to sulfoxide with trimethyl bromosilane .'- ¹² - 'The Nvoc groups remain on the peptide during the cleavage with TFA and the subsequent work-up. In this way, the amino groups that are to be freely available after the labeling reaction remain protected. The amino group to be modified is now freely available. The peptide can now be analyzed using standard techniques and further purified. The analysis was carried out using HPLC (RP18 column with gradient elution: solvent A = 0.1% TFA in distilled water and solvent B = 0.08% TFA in ACN) and electrospray mass spectroscopy (ESI-MS). Despite a certain stability of the Nvoc group against daylight and room lighting, it is advisable to darken the reaction vessels and avoid direct sunlight.

Example 2:

Synthesis of peptides using amino acids that contain photolabile protecting groups in their side chains

The peptide is synthesized by means of solid phase peptide synthesis with the orthogonal Fmoc / tert-butyl protective group strategy on a milligram scale (13.5 μmol) .'- ⁵ - ¹ For the ε-amino groups of Lys side chains, which are still protected during the labeling reaction and later biological examinations are to be freely available, only lysine derivatives with photolabile protected amino functions are used in the synthesis. These lysine derivatives are not commercially available and are therefore shown separately before the start of the synthesis. The implementation of the synthesis of an N ^ε -Nvoc-protected lysine derivative is described in the literature (Rusiecki, Bioorganic & Medicinal Chemistry Letters 1993). After synthesis, such peptides only contain a free amino group in the form of the N-terminus, which can then be selectively labeled.

Example 3:

Synthesis of peptides using amino acids whose side chains contain photolabile protective groups and whose N-terminal amino acid is protected at the N ^α - photolabile.

The peptide is synthesized as described in Example 2, but the labeling takes place on a Lys side chain. For this purpose, the lysine residue to be labeled is not used in a photolabile manner but in an acid-labile manner. At positions with lysine residues that are not to be modified during the labeling reaction, N ^ε -photolably protected lysine derivatives are used, as described in Example 2. To protect the N-terminus, however, an N ^α -photolabile-protected amino acid is used in the last coupling step of the peptide synthesis (position 1 in the peptide sequence). This N ^α -photolably protected amino acid is synthesized analogously to N ^ε -photolably protected lysine (Vossmeyer: Journal of applied physics 1998). Here are some more practical examples. Because of the clear advantages and greater flexibility, the method presented in exemplary embodiment 1 was used.

Embodiment 4: Representation of ³ H-propionyl-Lys ⁴ -NPY ( ³ H-NPY)

The neuropeptide Y (NPY) consists of 36 amino acids and contains a free amino group in the Lys side chain in addition to the N-terminus. ¹ - ^5] It could be shown that a free N-Teminus is required for full biological activity. In contrast, the amino function of the Lys side chain can be modified without loss of affinity for the display of radioligands. ^] These are commercially available in the form of ¹²³ I-NPY and H-NPY (Amersham).

To validate the Nvoc-based labeling method presented here and to compare it with previously known biological data of ³ H-NPY, N-terminal Nvoc-protected NPY was therefore first synthesized and then on the Lys ⁴ side chain by reaction with ³ H-propionyl -NHS ester radiolabelled. After photochemical cleavage of the Nvoc group and purification by HPLC, the H-NPY thus represented was compared in binding assays with the commercially available H-NPY.

a) Synthesis of N ^α -Nvoc-protected NPY (Nvoc-NPY)

NPY (MW = 4253.7) was generated using automated solid phase peptide synthesis. Following the synthesis, the free N-terminus of the peptide on the polymeric support was protected with the photolabile Nvoc protective group. The peptide was subsequently cleaved from the resin. The acid-labile protective groups were also removed. The crude peptide thus obtained was analyzed after working up and purified by means of preparative HPLC (FIG. 4). The presence of the Nvoc protective group can be recognized in the DAD spectrum by absorption at 350 nm (FIG. 5). The calculated molecular mass of Nvoc-NPY (MW = 4492.9) could be confirmed by the ESI-MS analysis shown in FIG. 6: m / z = 749.9 [M + 6H] ⁶⁺ (calculated: 749.8 ), 899.7 [M + 5H] ⁵⁺ (calculated: 899.6), 1123.8 [M + 4H] ⁴⁺ (calculated: 1124.2) and 1499.4 [M + 3H] ³⁺ (calculated : 1498.6). After cleaning, the desired product was homogeneous and with a purity of> 99%. b) ³ H labeling of N ^α -Nvoc-NPY, Nvoc cleavage and purification

The free amino group of the Lys ⁴ side chain of Nvoc-NPY (9 μg, 2 nmol) was derivatized by reaction with 100 μl N-succinimidyl [2,3-H] propionate (1 nmol, 3.7 MBq). A peak (R _t = 32.7 min, fractions 31-36) was isolated after irradiation with UV light and subsequent chromatographic separation of the unlabeled excess starting compound (whose retention time after Nvoc cleavage again corresponded to that of the unmodified NPY) , the retention time of which coincided with that of "cold" -propionylated NPY (FIG. 7). The NPY on Lys - "cold" - propionylated was previously after synthesis of Lys ⁴ (Dde) -NPY, cleavage of the Dde protective group and by subsequent Obtained reaction with propionic anhydride on the resin.

The radioactivity profile in the β-counter was determined by ³ H measurement of aliquots of the collected HPLC fractions. A peak can be seen in the HPLC chromatogram and in the radioactivity profile in fractions 31-36, which contained 0.8 MBq of radioactivity (22%) and thus corresponds to the desired radioactively labeled peptide (FIG. 8). It can therefore be assumed that it is H-propionyl-Lys ⁴ -NPY.

In competitive binding studies on the Y receptors Y1, Y2 and Y5, identical behavior was found in comparison to the commercially available ³ H-NPY, which was also marked at this position (FIG. 9). The binding assays were carried out as previously described. ^[5]

Embodiment 5: Representation of ³ H-propionyl-Lys ¹³ -PTH (1-34) -amide ( ³ H-PTH)

The parathyroid hormone (PTH) (1-34) amide (MW = 4116.8) contains three other amino groups in free form in addition to its N-terminus in Lys side chains (Lys ¹³ , Lys ²⁶ and Lys ²⁷ ). Because the N-terminus and the C-terminal region are thought to be important for binding to the PTH receptor, Lys was selected for labeling. ' ¹⁷¹ a) Synthesis of N ° and Lys ²⁶ ^'27 -Nvoc-protected PTH (1-34) -amide (Nvoc-PTH)

The peptide was synthesized using automated solid phase peptide synthesis. Protected Lys derivatives were used for positions 26 and 27 Dde side chains. After the Dde protecting groups had been split off, the Lys ²⁶ ^'27 amino groups and the N terminus were newly protected simultaneously with the Nvoc group. After splitting off and purification, a homogeneous product with a purity of> 95% was present. (Fig. 10) The presence of three Nvoc groups in the peptide (MW = 4836.4) could be confirmed by the DAD spectrum and ESI-MS: m / z = 807.4 [M + 6H] ⁶⁺ (calculated : 807.1), 968.6 [M + 5H] ⁵⁺ (calculated: 968.3) and 1209.6 [M + 4H] ⁴⁺ (calculated: 1210.1). (Fig. 11, 12)

b) ³ H-labeling of N ^α - and amide Lys ^26,27 -Nvoc-protected PTH (l-34), Nvoc-

Splitting off and cleaning

The free amino group of the Lys ¹³ side chain of Nvoc-PTH (1-34) amide (9.7 μg, 2 nmol) was treated with N-succinimidyl [2,3-H] propionate in borate buffer (pH = 8, 0) implemented. After irradiation with UV light and subsequent chromatographic separation of the unmarked excess starting compound (whose retention time of 17.3 min after the Nvoc cleavage again corresponded to that of the unmodified PTH (1-34) amide), a peak (R _t = 19 , 8 min, fraction 19-23), the retention time of which matched that of "cold" propionylated Lys ¹³ -PTH (1-34) amide (FIG. 13). Propionylated PTH (1-34) amide was previously obtained after synthesis of Lys ¹³ (Dde) -PTH (1-34) amide, cleavage of the Dde protective group and subsequent reaction with propionic anhydride on the resin. The radioactivity profile of the collected HPLC fractions was determined by ³ H measurement of aliquots in the ß counter. In the HPLC chromatogram and in the radioactivity profile, a peak can be seen in fractions 19-23 which contained 58.9 ^' kBq of radioactivity and corresponds to the desired radioactively labeled peptide. (Fig. 14) It can therefore be assumed that this is ³ H-propionyl-Lys ¹³ - PTH (1-34) amide.

In a competitive binding study on the PTH 1 receptor, an ICso value of 3.3 nM at a radioligand concentration of 1 nM showed almost identical behavior towards unlabeled PTH (1-34) amide (Fig. 15). The The binding assays were carried out as previously described. This showed that the PTH (1-34) amide marked at position 13 was selectively marked, is bioactive and can be used as a radioligand.

Embodiment 6: Representation of N-terminal Nvoc-protected Lys -hPP

The human sequence of the pancreatic polypeptide (hPP) has an amino group only through the N-terminus. Since PP and NPY belong to the same family of peptides, the N-terminus of PP is also considered to be of particular importance in binding to the Y4 receptor. When designing a radio ligand, it should therefore be unmodified. For this reason, a Lys residue was introduced into the non-conserved position 4 during peptide synthesis. Later radioactive marking is to take place there.

a) Synthesis of N ^α -Nvoc-protected Lys ⁴ -hPP (Nvoc-hPP)

After the synthesis of Lys-hPP (MW = 4180.9), the N-terminal amino group of the peptide still attached to the resin was newly protected by the Nvoc group. After splitting off, working up and cleaning by means of preparative HPLC, a homogeneous product with a purity of>95%> was present. (Fig. 16) The presence of the Nvoc group in the peptide (MW = 4421.1) could be confirmed by the absorption at 350 nm in the DAD spectrum and ESI-MS: m / z = 738.5 [M + 6H] ⁶⁺ (calculated: 737.9), 885.6 [M + 5H] ⁵⁺ (calculated: 885.2), 1106.3 [M + 4H] ⁴⁺ (calculated: 1106.3), 1474.0 [ M + 3H] ³⁺ (calculated: 1474.7) and 2212.4 [M + 2H] ²⁺ (calculated: 2211.6). (Fig. 17, 18)

The invention and its exemplary embodiments are explained in more detail by the following figures, which show:

Fig. 1: Schematic representation of the synthesis of Nvoc-protected peptides with a free Lys side chain

Fig. 2: Schematic representation of the synthesis of Nvoc-protected peptides with a free N-terminus

Fig. 3: Schematic representation of the selective radioactive labeling of a

Lys side chain of an Nvoc-protected peptide and subsequent Nvoc cleavage

Fig. 4: HPLC chromatogram of N-terminal Nvoc-protected NPY

(220 nm; R _t = 21.3 min; purity> 99%)

5: DAD spectrum of the HPLC chromatogram of N-terminal Nvoc-protected NPY from FIG. 4. The DAD spectrum of 200-400 nm is shown in the lower part, the absorption of the peptide bonds at 220 nm is shown in the upper part (top line) and the absorption Nvoc group at 350 nm (bottom line).

Fig. 6: ESI-MS of N-terminal Nvoc-protected NPY.

Fig. 7: HPLC chromatogram of the ³ H labeling approach of Nvoc-NPY after UV irradiation. The peak with the retention time of 30.3 min corresponds to unlabelled NPY after the Nvoc cleavage and the peak at 32.7 min corresponds to ³ H-propionyl-Lys ⁴ -NPY.

Fig. 8: Radioactivity profile of the after labeling, irradiation and HPLC

Nvoc-NPY fractions obtained (fractions 20-40; dead volume from the detector to the fraction collector approx. 1 min).

Fig. 9: Binding curves of the ³ H-propionyl obtained according to the Nvoc strategy

Lys ⁴ -NPY with unlabelled NPY as competitor. The displacement curves at A) Yl receptor, B) Y2 receptor and C) Y5 receptor are shown. The one with a radioligand concentration of! IC ₅₀ values obtained in the range from 1 to 5 nM and thus, like the commercially available ³ H-NPY, indicate an almost identical binding behavior of the radioligand and competitor:

Fig. 10: HPLC chromatogram (220 nm) of N-terminal and Lys' Nvoc-protected PTH (1-34) amide. (R _t = 23.8 min, purity> 95%)

Fig. 11: DAD spectrum of the HPLC chromatogram of N-terminal and

Lys ²⁶ ^'27 Nvoc-protected PTH (l-34) amide in Fig. 10. In the lower part of the spectrum is shown DAD 200-400 nm, at the top of the absorption of the peptide bonds at 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (bottom line). The presence of three Nvoc groups in the molecule results in a stronger absorption for this group compared to FIG. 5 at the same concentrations.

Fig. 12: ESI-MS of N-terminal and Lys ²⁶ ^'27 Nvoc-protected PTH (l-34) - amide.

Fig. 13: HPLC chromatogram of the ³ H labeling batch of Nvoc-PTH (1-

34) -amide after UV radiation. The peak at 17.2 min corresponds to the unlabeled PTH (1-34) amide after the Nvoc cleavage due to its retention time and the peak at 19.8 min corresponds to ³ H-propionyl-Lys ¹³ -PTH (1-34) - amide.

Fig. 14: Radioactivity profile of the after labeling, irradiation and HPLC

Nvoc-PTH (1-34) -amide fractions obtained (fractions 10-27; dead volume from the detector to the fraction collector approx. 1 min).

15: Binding curve of the H-propionyl obtained according to the Nvoc strategy

1 ^

Lys -PTH (1-34) amide on the PTHI receptor with unlabeled PTH (1-34) amide as competitor. The IC 50 value of 3.3 nM obtained at a radioligand concentration of 1 nM indicates an almost identical binding behavior of the radioligand and competitor.

Fig. 16: HPLC chromatogram (220 nm) of N-terminal Nvoc-protected

Lys ⁴ -hPP. (R _t = 24.5 min, purity> 95%) FIG. 17: DAD spectrum of the HPLC chromatogram of N-terminal Nvoc-protected Lys-hPP from FIG. 16. The DAD spectrum of 200-400 nm is shown in the lower part, the absorption of the peptide bonds is shown in the upper part 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (lower line) are shown.

Figure 18: ESI-MS of N ^α -Nvoc-Lys ⁴ -hPP.

Bibliography :

[I] Hulme, E.C., Receptor-Ligand Interactions: A Practical Approach. 1992, Oxford: IRL Oxford University Press.

[2] Rehm, H., The Experimentator: Protein Biochemistry / Proteomics .. Vol. 3. 2000,

Heidelberg: Spectrum publishing house.

[3] Bolton, A.E. and Hunter, W.M., Labeling of proteins to high specific radioactivities by conjugation to an iodine-125-containing acylating agent. Application to the radiomunoassay. Biochem. J., (1973) 133, 529-538

[4] Vincent, J.P. and Gaudriault, G., The importance of pH in labeling proteins and peptides by acylation of their a-amino groups with a reactant carrying an activated carboxylic group. (1993) in FR 2687680: France.

[5] Cabrele, C, Wieland, H.A., Koglin, N., Stidsen, C. and Beck-Sickinger, A.G.,

Ala (31) -Aib (32): identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y (5) -receptor. Biochemistry, (2002) 41, 8043-9.

[6] Pillai, V.N.R., Photoremovable protecting groups in organic synthesis. Synthesis,

(1980) 7, 1-25

[7] Dorman, G. and Prestwich, G.D., Using photolabile ligands in drug discovery and development. Trends Biotechnol, (2000) 18, 64-77.

[8] Fodor, S.P., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T. and Solas, D., Light-directed, spatially addressable parallel chemical synthesis. Science, (1991) 251, 161-13.

[9] Bycroft, B.W., Chan, W.C., Chhabra, S.R., Teesdale-Spittle, P.H. and Hardy,

P.M., A novel amino protection-deprotection procedure and its application in solid phase peptide synthesis. J. Chem. Soc, Chem. Commun. , (1993) 9, 116-111

[10] Rusiecki, V.K. and Warne, S.A., Synthesis of Na-Fmoc-Ne-Nvoc-lysine and use in the preparation of selectively functionalized peptides. Bioorg. Med. Chem. Lett.,

(1993) 3, 707-710

[I I] Kaiser, E., Colescott, R.L., Bossinger, CD. and Cook, P.I., Color test for detection of free terminal amino groups in the solid phase synthesis of peptides. Anal. Biochem., (1970) 3- /, 595-598

[12] Beck, W. and Jung, G., Convenient reduction of S-oxides in synthetic peptides, lipopeptides and peptide libraries. Lett. Pept. Sci., (1994) 1, 31-37 [13] Stefanowicz, P. and Siemion, LZ., Reactivity of N-hydroxysuccinimide esters. Pole. J. Chem., (1992) 66, 111-118

[14] Tang, Y.S., Davis, A.M. and Kitcher, J.P., N-succinimidyl propionate: characterization and optimum conditions for use as a tritium labeling reagent for proteins. J. Labeled Compd. Radiopharm., (1983) 20, 277-284

[15] Cabrele, C. and Beck-Sickinger, A.G., Molecular Characterization of the Ligand-Receptor Interaction of the Neuropeptide Y Family. J. Peptide Sci., (2000) 6, 97-122.

[16] Chang, R.S., Lotti, V.J., Chen, T.B., Cerino, DJ. and Kling, P.J., Neuropeptide Y (NPY) binding sites in rat brain labeled with 1251-Bolton-Hunter NPY: comparative potendies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens. Life Sciences, (1985) 37, 2111-2122

[17] Juppner, H., Schipani, E., Bringhurst, FR, McClure, L, Keutmann, HT, Potts, JT, Kronenberg, HM, Abou-Samra, AB, Segre, GV and Gardella, TJ, The ex- tracellular amino-terminal region of the parathyroid hormone (PTH) / PTH-related peptide reeeptor determines the binding affinity for carboxyl-terminal fragments of PTH- (1- 34). Endocrinology, (1994) 134, 879-884

List of abbreviations

The following abbreviations are used in the text and in the figures:

2-C1-Z-2-chlorobenzyloxycarbonyl

ACN acetonitrile

Boc- tert-butoxycarbonyl-

Bzl Benzyl

DAD diode array detector

DCM dichloromethane

Dde- l- (4,4-dimethyl-2,6-dioxocyclohex-l-ylidenes) ethyl

DIC N, N-di-isopropyl-carbo-di-imide

DIPEA N, N-di-isopropyl-ethyl-amine

DMF N, N-dimethylformamide

ESI-MS electrospray ionization mass spectrometry

Fmoc- (9-fluoro-enyl) methoxycarbonyl-bound

H tritium

HF hydrogen fluoride, hydrofluoric acid

HOBt 1-hydroxy-benzotriazole

HPLC High Performance Liquid Chromatography ivDde- l- (4, 4-dimethyl-2,6-dioxocyclohex-l-ylidene) -3-methylbutyl-

K lysine

Bq Becquerel m mass

Mmt 4-methoxytrityl

Mtt 4-methyltrityl

NHS N-hydroxysuccinimide

N ^α nitrogen of the α-amino group

N ^ε nitrogen of the ε-amino group (e.g. in lysine)

NPY neuropeptide Y

Nvoc nitroveratryloxycarbonyl P proline

PP pancreatic polypeptide

PTH para-thyroid hormone

S serine

SG protection group

T polymeric carrier

TIS triisopropylsilane tBu: tert-butyl

TFA trifluoroacetic acid

Tfa trifluoroacetyl

UV ultraviolet

X any amino acid

Y tyrosine

Z- benzyloxycarbonyl

The three- and one-letter codes of the amino acids used correspond to the proposals of the IUPAC-IUB Commission for biochemical nomenclature.

Claims

claims

1. A method for the selective labeling of amino groups in peptides, wherein a peptide is built up by chemical solid-phase peptide synthesis from amino acid components on a support material, characterized in that a.) In the peptide synthesis, an amino acid component is incorporated at at least one position to be labeled carries an amino group which is protected by a group which differs functionally from the protective groups on the amino groups which are not to be labeled, b.) after the peptide synthesis, selectively removes the protective group at the position to be labeled and the peptide is detached from the support material, c .) a mark is introduced into the selectively deprotected peptide thus obtained in solution at the position to be labeled via amino-reactive substances, d.) the protective groups on the amino groups which are not to be labeled are subsequently cleaved off and the selectively labeled peptide thus obtained is purified in a known manner becomes.

2. The method according to claim 1, characterized in that in the peptide synthesis, the amino groups not to be labeled carry photolabile protective groups. '

3. The method according to claim 1, characterized in that in the peptide synthesis, the amino groups not to be labeled, initially carry hydrazine-labile protective groups or protective groups which can be split off with a dilute acid, such as 1% by volume trifluoroacetic acid (TFA) in dichloromethane (DCM) ,

4. The method according to claim 3, characterized in that the protective groups of all amino groups which are not to be labeled are selectively removed after the peptide synthesis and before the peptide is detached from the support material and the amino groups which are not to be labeled are newly protected with photolabile protective groups and the peptide is then subsequently is detached from the solid support material.

5. The method according to claim 1, characterized in that 'the amino acid building blocks used in the peptide synthesis on the non-labeled amino groups, base-labile protective groups, preferably 9-fluoro-enyl) methoxycarbonyl (Fmoc) groups, after installation each amino acid component that is not to be labeled, the base-labile protective group is split off and the amino group that is not to be labeled is newly protected with photolabile protective groups.

6. The method according to any one of claims 1 to 5, characterized in that in the synthesis, the amino groups to be labeled carry protective groups, preferably tert-butoxycarbonyl (Boc) groups, which do not contain dilute acids such as 1% by volume TFA in DCM, but can be split off at high acid concentrations, such as over 50 vol% TFA in DCM.

7. The method according to any one of claims 2 to 4, characterized in that the photolabile protecting groups are of the 20-nitrobenzyl type, preferably nitroveratryloxycarbonyl (Nvoc) groups.

8. The method according to claim 3, characterized in that the hydrazine-labile protective groups dioxocyclohexylidene compounds, preferably 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) or 1- (4th , 4-dimethyl-2,6-dioxocyclohex-l-ylidene) -3-methylbutyl (ivDde) groups.

, A method according to claim 3, characterized in that the protecting groups which can be split off with a dilute acid are trityl compounds, preferably 4-methoxytrityl (Mmt) or 4-methyltrityl (Mtt) groups.

10. The method according to any one of claims 1 to 9, characterized in that the label is a radioactive isotope.

11. The method according to any one of claims 1 to 10, characterized in that N-hydroxysuccinimide (NHS) esters are used as amino-reactive substances.

12. The method according to claim 11, characterized in that a radioactive NHS ester, preferably N-succinimidyl- [2,3- ³ H] - propionate, is used for labeling.

13. Use of peptides in which all amino groups which are not to be labeled are protected by protective groups, preferably photolabile protective groups, and at least one amino group to be labeled is unprotected for the production of a selectively radioactively labeled peptide.