DE10334499A1 - Synthesis of peptides labeled selectively on amino groups, useful in biotechnology and diagnosis, uses selectively removable protecting group at labeling position - Google Patents

Abstract

Method for selectively labeling amino groups in peptides (I), prepared by chemical solid-phase synthesis. Method for selectively labeling amino groups in peptides (I), prepared by chemical solid-phase synthesis comprises (1) incorporating, during synthesis and at the positions intended for labeling, an amino acid synthon in which amino is protected by a group (PG1) that is functionally different from the group (PG2) on amino groups that are not intended for labeling; (2) at the end of synthesis, selective removal of PG1 and release of (I) from its carrier; (3) reacting the released peptide, in solution, with a labeling reagent, specifically at free amino; and (4) removal of PG2 and conventional purification of the product.

Description

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 radioactively labeled and thus easily detectable peptides. However, this label must not restrict biological activity. In this case, radioactively labeled peptides have z. 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]

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 ( ¹²⁵ 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. ³⁵ 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) and reagents which 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 ester radioactive acyl residues are commercially available, such. B. 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.

(Formel 1)

(Formula 1)

• a.) mit einem nach der Synthese noch am Trägermaterial gebundenen und bis auf die zu markierende Stelle seitenkettengeschützten Peptid oder
• b.) auch mit einem freien Peptid in Lösung erfolgen.

Indirect labeling of peptides can either

• 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.) also with a free peptide in solution.

The advantage of radioactive labeling one on the carrier material (Resin) bound peptide consists in the possibility of amino acid side chains to protect selectively and derivatize. However, this advantage will follow the synthesis by e.g. T. complex work steps under radioactive Conditions lost. adversely here is that with resin-bound peptides only with approaches the milligram range can be worked. Therefore requires one radioactive labeling on the resin causes and causes considerable amounts of radioactivity very high cost.

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 in the case of peptide sequences that are difficult to synthesize and thus more contaminated raw peptides, a large part of the radioactivity used passes through likewise labeled during the processing Missing sequences are lost again. 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.

For however, numerous purposes are usually just a few micrograms high affinity radioactive labeled peptide sufficient. Farther are used in screening tests frequently different peptides marked at defined positions needed in new receptor-ligand interaction studies. Most of time so it makes sense first display only small amounts of a radioactive biomolecule for testing and deploy.

With indirect marking in solution is the peptide according to the prior art following the peptide synthesis radioactively marked after removal of all protective groups.

This also has advantages and disadvantages. The advantage is that even very small quantities (μg range) can be handled are. A selective labeling of individual amino acids is, however, as soon as the peptide has several reactive groups of the same type (e.g. several Contains amino groups), impossible. With longer ones This is common for peptides the case.

A selective marking is u. a. necessary, if single reactive groups for the full biological activity of the peptide must not be modified.

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

A chemical distinction between two lysines is impossible: An attempt to chemically differentiate between the 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.

Object of the present invention is to provide a method for selectively labeling peptides. This procedure is intended in particular to be the selective step radioactive labeling of the peptides in solution may be possible. The peptides are said to in addition to the amino group to be labeled, also other amino groups can have. In particular, it should be possible with the invention, peptides regardless of their sequence selective from the microgram (μg) scale radioactive mark.

• 1. bei der Peptidsynthese, an der jeweils zu markierenden Position ein Aminosäurebaustein eingebaut wird, der eine Aminogruppe trägt, die durch eine Gruppe geschützt wird, die sich funktionell von den Schutzgruppen an den nicht zu markierenden Aminogruppen unterscheidet;
• 2. im Anschluss an die Peptidsynthese die Schutzgruppe an der zu markierenden Position selektiv entfernt und das Peptid vom Trägermaterial gelöst,
• 3. in das so erhaltene selektiv-entschützte Peptid in Lösung über aminoreaktive Substanzen an der zu markierenden Position eine radioaktive oder mit anderen Methoden detektierbare Markierung eingeführt wird, und
• 4. anschließend die Schutzgruppen an den nicht zu markierenden Aminogruppen abgespalten werden und das so erhaltene selektiv markierte Peptid in bekannter Weise gereinigt wird.

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 is advantageous with just a few μg peptide manageable and is not tied to methodological minimum quantities.

The following for the labeling of peptides Methods described with radioactive isotopes can be also for insertion use other markers that are detectable with other methods.

With the method according to the invention homogeneous labeled peptides are obtained in a quality that meet the high requirements for correspond to quantitative biological studies. As a result of this already on a micro scale compatible method the amount of radioactivity to be used for radioactive labeling according to the desired Control amount of radio ligand. Another advantage of this method lies in the reduced number of labor-intensive radioactive work steps and in the associated with less equipment and analytical effort feasibility a selective marker. This can make this method cheaper and easier to do be than previous methods.

The amino group to be labeled selectively can advantageously be chosen freely become, d. H. it is either an amino group in the side chain of an amino acid building block (e.g. a ε group of a lysine) or the N-terminus of the assembled peptide. Also selective labeling to several amino groups is 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 amide or peptide bonds. In particular the embodiments of the peptide synthesis are introduced at the C-terminus of the peptide an amino group (NH ₂ ) as the C-terminal amide or a substituted amino group (NHR, R = alkyl) or an alcohol residue. 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), a peptide is built up sequentially, in which one amino acid building block per synthesis step is added to the free N-terminus of the growing peptide chain is added to the C-terminus. 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 ⁽⁵⁾ .

So only in peptide synthesis the free N-terminus on the carrier bound amino acid with the next one amino acid building block will be responded to the synthesis of amino acid building blocks used in which except the C-terminus all chemically reactive Groups of amino acids, like the α-amino group and other functional groups in the side chains such as amino groups (Lysine), or e.g. B. free hydroxyl groups (tyrosine, serine) with corresponding Protection groups are provided.

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

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

The Boc / Bzl strategy is through one graded acid lability marked. The α-amino groups are acid-labile tert-butoxycarbonyl groups (Boc groups) protected. While The synthesis is here to generate a new free N-terminus the Boc group on the α-amino group usually split off 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 by benzyl (Bzl) groups or benzyloxycarbonyl (Z) groups protected, the z. B. only by treatment with the stronger hydrofluoric acid (HF) can be removed again.

In the orthogonal Fmoc / ^t 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 usually protected by acid-labile groups.

In this strategy, hydroxyl groups are mostly protected by tertiary butyl groups ( ^t 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).

Peptide synthesis is preferably carried out according to the orthogonal Fmoc / tert-butyl protective group strategy. ^[5]

According to the invention during the peptide synthesis at least one later an amino acid building block to be marked, which carries an amino group, protected by a group that is functional from the protecting groups to the not distinguishes distinctive amino groups. In addition, the protection group functional of the protecting groups on the amino groups which the Form peptide bond, differentiate.

Because of this functional difference will it be possible the protective group at the position to be marked in another To selectively remove step of the process.

This functional difference is based on different chemical properties, such as durability across from certain chemical substances.

In the method according to the invention, the one to be marked later is used Amino group first while The synthesis is protected by a group that deals with a method split off, in which the amino protecting groups on the amino groups not to be labeled remain.

In the process 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 by a chemically labile protective group (SG) B. a benzyloxycarbonyl (Z group in the case of the Boc / Bzl strategy) or a Boc group (in the case of the Fmoc / ^t Bu strategy).

In one embodiment of the method this is achieved in that the peptide synthesis does not to be labeled amino groups photolabile protecting groups, e.g. B. Nitroveratryloxycarbonyl (Nvoc) become. This is achieved by installing appropriately protected amino acid building blocks, z. B. Lysine with Nvoc-protected Side chain, reached.

Lysines that are protected on the side chain with Nvoc (N ^ε -Nvoc-lysines) are e.g. B. after implementation obtained from N ^α -protected lysines (e.g. N ^α -Boc-Lys or N ^α -Fmoc-Lys) with Nvoc chloride ^[10] .

Should be the N-terminal amino group are selectively labeled, the N-terminal amino group that follows the last coupling step of the synthesis is initially still protected, deprotected and the peptide from the support material split off while all other amino groups not to be marked in the side chains, are derivatized with a photolabile protecting group.

In the event that the N-terminal amino group is not to be selectively labeled, an N ^α -photolably-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).

(Formel 2) The photolabile protective groups used in the process according to the invention are preferably groups of the ortho-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. ^{[6-8 ]} . Under the influence of light, compounds of the ortho-nitrobenzyl type undergo photoenolization and fragmentation to give ortho-nitrosobenzaldehydes. The lamp strength can vary within a 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 / ^t 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 / ^t Bu strategy), the peptide is simultaneously detached from the solid phase support and further protective groups are removed ,

The photolabile protecting groups the remaining amino groups that are not to be labeled are included not split off.

The peptide in solution now only contains at the position to be marked a free amino group that is unprotected and can be marked selectively. This selective marking happens like later Move closer explains 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 3 shown.

The direct introduction of amino acid building blocks with photolabile protecting groups during of peptide synthesis, however, has the disadvantage that the entire Synthesis must be done in the dark. Another disadvantage is that Amino acids protected with photolabile protecting groups are not yet commercially available available are.

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 1 shown.

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

In this embodiment, all amino groups which are preferred during peptide synthesis should not be labeled with a hydrazine-labile protective group, particularly preferably with 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl- (Dde) or 1- (4,4-dimethyl-2,6- dioxocyclohex-1-yliden) -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. appropriately protected amino acid building blocks used. In these amino acid building blocks, such as. B. a lysine protected on the ε-amino group with Dde (N ^α -Fmoc-N ^ε -Dde-lysine), the amino group, which should not be labeled, is protected with a hydrazine-labile 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 / ^t 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 / ^t Bu strategy, an amino acid building block 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, it contains selective amino acid building block to be marked correspondingly one with a base-labile protective group (e.g. Fmoc) protected Amino group.

In the event that 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 / ^t Bu strategy, the α-amino group (N ^α ) with an acid-labile (in hydrazine-stable) protective group (e.g. B. Boc) is protected. This variant is shown schematically in 2 shown. 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. In the case of the Fmoc / ^t Bu strategy, this is a base-labile protecting 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. z. B removed with 2% hydrazine in DMF ^[9] .

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

These protecting groups are instead with hydrazine with a diluted Acid within selectively split off from 4 by 2 minutes, e.g. B. with 1% TFA in dichloromethane (DCM). The dilute acid, in this case a scavenger, is preferably preferred 1% to 5% TIS (triisopropylsilane) added.

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

By the selective separation of the with hydrazine or protective groups which can be split off under weakly acidic conditions all amino groups not to be labeled are now freely available. Just the amino group to be labeled is protected. The peptide is still to the carrier material bound.

All freely available amino groups are now preferred simultaneously with a photolabile protecting group of the ortho-nitrobenzyl type, such as. B. Nvoc provided.

For this purpose, an activated derivative of the photolabile protective group (e.g. Nvoc chloride), N-hydroxybenzotriazole (HOBt, Novabiochem) and diisopropylethylamine (DIPEA, Fluka, Buchs, Switzerland) and 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 approximately 0.5 ml to 1 ml. The reagents, which have also been swollen in DMF, are added to the reagents, which are also dissolved in DMF, and left for at least 3 h 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. ^[11] 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. ^[10] This variant also advantageously does not require the use of special Nvoc-derivatized amino acids, which are not commercially available.

After all amino groups 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 / ^t Bu strategy, the acid-labile protective 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 only contains a free amino group at the position to be marked that is unprotected, and can be marked selectively. The photolabile protecting groups on the remaining amino groups, following the labeling easy to remove by UV light.

In the case of the Fmoc / ^t 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.

Because the base-labile Fmoc group is not is particularly stable, this embodiment is only suitable for the construction of short peptides with the Boc / Bzl strategy.

In an alternative variant of the Invention is therefore during the synthesis immediately after incorporation of the relevant amino acid and deprotection the amino function in question on the amino acid on the amino groups photolabile protecting group introduced.

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

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 reaction 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, synthesis is at the end all amino groups not to be labeled in the peptide built up protected by a photolabile group (such as Nvoc).

That after the separation from the polymer carrier in solution contains peptide now a free amino group only at the position to be marked, the unprotected is present and can be marked selectively. The photolabile protecting groups on the remaining amino groups can be easily done after the labeling remove by UV light.

The peptide in which one of the The previously described variants of the method according to the invention are those to be marked Selectively deprotected amino group is in solution as follows marked with an amino reactive substance.

As amino reactive substances preferably acylating reagents, such as N-hydroxysuccinimide (NHS) esters, are used. These NHS esters, as described schematically in Formula 1, an acyl group containing the corresponding marker from Transfer NHS ester to the amino group.

As a marker, the acyl group preferably contains radioactive isotopes, such as. B. ¹²⁵ iodine ( ¹²⁵ I), ³⁵ sulfur ( ³⁵ S) and tritium ( ³ H).

In other embodiments, the acyl group other markers tied like that over the affinity biotin detectable to streptavidin / avidin, or immunological detectable haptens, such as. B. digoxygenin or fluorescein.

Fluorescence-detectable dyes can also be used as markers, whereby when using photolabile protective groups in the process according to the invention, according to the The protective group is split off at a wavelength, where the dyes are not bleached.

The labeling reaction can already with a few micrograms of peptide. Since these are minor It was therefore difficult to weigh quantities, which is why the with preparative HPLC cleaned and photolabile protected Peptide made in double distilled water, the desired amount (e.g. 2 nmol, depending on the molecular weight of the peptide approx. 10 μg) from the stock solution removed 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. 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 marking, the photolabile Protecting groups on the amino groups not to be labeled under the described conditions separated by means of UV light in an aqueous medium.

There is a previous one in the watery environment It is not necessary to trap unreacted esters. After the response time in watery Milieu can be assumed that the ester aminolyses was completely hydrolyzed or by the side reaction with water is present and therefore no longer with the newly released amino groups able to react. After a labeling reaction in DMF should however, a nucleophile like glycine can be added to make it unselective Markings by unreacted esters after the to avoid photolabile protecting group.

To a greater degree of fragmentation in particular of peptides containing Trp by the UV radiation should be avoided with these peptides the UV exposure is limited to a maximum of 30 min become. Here too, the photolabile protective group becomes almost quantitative removed from the peptide. To side reactions, especially education of covalent adducts of the split-off protective group with the released ones Avoiding amino groups will result in cleavage in an acidic environment carried out. For this purpose, the marking approach with a 0.1% aqueous TFA / ACN solution added. The ACN content can already affect the starting conditions below of the HPLC gradient can be set.

The peptide is then known Way cleaned. This is preferably done by means of HPLC (high Performance Liquid Chromatography). Since the marking is never 100% extends and also the Nvoc peptide in excess across from radioactive ester was used, a separation of the derivatized of the non-derivatized Peptide done. Because the two peptides are dependent the transferred Group mostly only marginally to differentiate in terms of their elution behavior became expedient for separation selected a gradient elution with two eluents. The Gradient is possible depending on the peptide sequence and the quality of the column used and may not be for everyone Sequences can be generalized. The eluent used are: 0.08% TFA in acetonitrile and 0.1% TFA in water.

Detailed descriptions of the HPLC of peptides give the following reference: Hancock, W. S .; Sparrow, J. T .: HPLC Analysis of biuological compound, Vol 26, 1st edition, Marcel Dekker Inc., New York. 1984th

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

Through the following examples the invention becomes closer explains:

Example 1:

Synthesis of Lys (Dde) protected peptides and replacement of the Dde protective group by photolabile which can easily be split off in solution protecting groups

The peptide is first synthesized using solid phase peptide synthesis using the orthogonal Fmoc / tert-butyl protective group strategy on a milligram scale (13.5 μmol) ^[5] . 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- 1-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 amino group to be modified later, a certain Lys side chain or the N-terminus, then remains protected with the acid-labile Boc protective 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. On the other hand, if 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 the Dde protecting group (s) had been split off then provide all free amino groups with the photolabile Nvoc protective group at the same time.

The photolabile Nvoc group was installed on the resin at the positions that should be free again after the radioactive labeling. For this purpose, 5 equivalents of 6-nitroveratryloxycarbonyl chloride (Nvoc-C1, Sigma-Aldrich), 5 equivalents of N-hydroxybenzotriazole (HOBt, Novabiochem) and 10 equivalents of diisopropylethylamine (DIPEA, Fluka) were used, based on the number of available free amino functions. 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 has ended, the KAISER test is used to check whether all the free amino groups have reacted. ^[11] 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). At the same time, the acid-labile side chain protective groups are removed. 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 trimethylbromosilane. ^[12]

The Nvoc groups remain during the Cleavage with TFA and subsequent processing on the peptide. In this way, the amino groups remain after the labeling reaction should be freely available, still protected. The one to be modified Amino group is now freely available. The peptide can now be processed using standard techniques analyzed and further cleaned. The analysis was carried out using HPLC (RP18 column with gradient elution: Solvent A = 0.1% TFA in aqua dest. and solvent B = 0.08% TFA in ACN) and electrospray mass spectroscopy (ESI-MS). Despite a certain stability towards the Nvoc group Daylight and room lighting make it advisable to darken the reaction vessels and to avoid direct sunlight.

Example 2:

Synthesis of peptides under Use of amino acids, which contain photolabile protective groups in their side chains

The peptide is synthesized using solid phase peptide synthesis using the orthogonal Fmoc / tert-butyl protecting group strategy on a milligram scale (13.5 μmol). ^[5] For the 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, only lysine derivatives with photolabily 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. which 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).

Below are some more practical examples listed. Because of clear advantages and greater flexibility that was in the embodiment 1 presented procedures applied.

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 ⁴ side chain in addition to the N-terminus. ^[5] It could be shown that a free N-teminus is required for full biological activity. The amino function of the Lys ⁴ side chain, on the other hand, can be modified without loss of affinity for the preparation of radioligands. ^[16] These are commercially available in the form of ¹²⁵ I-NPY and ³ 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 ³ H-NPY thus represented was compared in binding assays with the commercially available ³ 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 preparative HPLC ( 4 ). The presence of the Nvoc protective group can be recognized in the DAD spectrum by absorption at 350 nm ( 5 ). By in 6 ESI-MS analysis, the calculated molecular mass of Nvoc-NPY (MW = 4492.9) was confirmed: 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 purification, the desired product was therefore 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 Lys (ug 9, 2 nmol) ⁴ side chain of Nvoc-NPY was prepared by reaction with 100 ul N-succinimidyl [2,3- ³ H] propionate (1 nmol, 3.7 MBq) derivatized. 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), a peak (R _t = 32.7 min, fractions 31–36) was isolated, the Retention time coincided with that of "cold" propionylated NPY ( 7 ). The NPY "cold" -propionylated on Lys ⁴ was previously obtained after synthesis of Lys ⁴ (Dde) -NPY, cleavage of the Dde protective group and subsequent 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. In the HPLC chromatogram and in the radioactivity profile, a peak can be seen in fractions 31-36, which contained 0.8 MBq of radioactivity (22%) and thus corresponds to the desired radioactively labeled peptide ( 8th ). It can therefore be assumed that it is ³ 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 labeled at this position ( 9 ). The binding assays were carried out as previously described. ^[5]

Embodiment 5: Representation of ³ H-propionyl-Lys ¹³ -P7H (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 considered important for binding to the PTH receptor, Lys ¹³ was selected for labeling. ^[17]

a) Synthesis of N ^α and Lys ^26,27 -Nvoc-protected PTH (1-34) amide (PTH-Nvoc)

The peptide was synthesized using automated solid phase peptide synthesis. Protected Lys derivatives were used for positions 26 and 27 Dde side chains. After removal of the Dde protecting groups, the Lys ^26,27-amino groups and the N-terminus of newly protected simultaneously with the Nvoc group. After splitting off and purification, a homogeneous product with a purity of> 95% in front. ( 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). ( 11 . 12 )

b) ³ H-labeling of N ^α - and Lys ^26,27 -Nvoc-protected PTH (1-34) amide, Nvoc cleavage and purification

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- ³ H] propionate in borate buffer (pH = 8 , 0) implemented. After irradiation with UV light and subsequent chromatographic separation of the unlabeled 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 ( 13 ). The LTH ¹³ - "cold" propionylated PTH (1-34) amide was previously synthesized after synthesis of Lys ¹³ (Dde) -PTH (1-34) amide, cleavage of the Dde protecting group and subsequent reaction with propionic anhydride Received resin.

The radioactivity profile of the collected HPLC fractions was determined by ³ H measurement of aliquots in the β counter. A peak can be seen in the HPLC chromatogram and in the radioactivity profile in fractions 19-23, which contained 58.9 kBq of radioactivity and corresponds to the desired radioactively labeled peptide. ( 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 PTH1 receptor, an IC ₅₀ value of 3.3 nM at a radioligand concentration of 1 nM showed almost identical behavior towards unlabeled PTH (1-34) amide ( 15 ). The binding assays were carried out as previously described. ^[5] 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 ⁴ -hPP

The human sequence of the pancreatic Polypeptide (hPP) has an amino group only through the N-terminus. Because PP just as NPY belongs to the same peptide family, so does the N-terminus of PP is of particular importance in binding to the Y4 receptor awarded. Therefore, when designing a radio ligand, it should present unmodified. For this reason, during the Peptide synthesis a Lys residue in the non-conserved position 4 introduced. There should be a later one radioactive labeling.

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

After the synthesis of Lys ⁴ -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. ( 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). ( 17 . 18 The invention and its exemplary embodiments are explained in more detail by means of the following figures, which show:

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

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

3 : Schematic representation of the selective radioactive labeling of a Lys side chain of an Nvoc-protected peptide and subsequent Nvoc cleavage

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 from N-terminal Nvoc-protected NPY 4 , The lower part shows the DAD spectrum from 200–400 nm, the upper part shows the absorption of the peptide bonds at 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (lower line).

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

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.

8th : Radioactivity profile of those obtained after labeling, irradiation and HPLC from Nvoc-NPY Fractions (fractions 20–40; dead volume from detector to fraction collector approx. 1 min).

9 : Binding curves of the ³ H-propionyl-Lys ⁴ -NPY obtained according to the Nvoc strategy with unlabelled NPY as competitor. The displacement curves at A) Y1 receptor, B) Y2 receptor and C) Y5 receptor are shown. The IC ₅₀ values obtained at a radioligand concentration of 1 nM are in the range from 1 to 5 nM and thus, like the commercially available ³ H-NPY, refer to an almost identical binding behavior of the radioligand and competitor.

10 : HPLC chromatogram (220 nm) of N-terminal and Lys ^26.27 Nvoc-protected PTH (1-34) amide. (R _t = 23.8 min, purity> 95%)

11 : DAD spectrum of the HPLC chromatogram of N-terminal and Lys ^26.27 Nvoc-protected PTH (1-34) amide 10 , The lower part shows the DAD spectrum of 200–400 nm, the upper part shows the absorption of the peptide bonds at 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (lower line). The presence of three Nvoc groups in the molecule results in a stronger absorption compared to this group at the same concentrations 5 ,

12 : ESI-MS from N-terminal and Lys ^26.27 Nvoc-protected PTH (1-34) amide.

13 : HPLC chromatogram of the ³ H labeling approach of Nvoc-PTH (1-34) amide after UV irradiation. The peak at 17.2 min corresponds to the unlabeled PTH (1-34) amide after the Nvoc elimination due to its retention time and the peak at 19.8 min corresponds to ³ H-propionyl-Lys ¹³ -PTH (1-34) - amide.

14 : Radioactivity profile of the fractions obtained after labeling, irradiation and HPLC of Nvoc-PTH (1-34) amide (fractions 10-27; dead volume from the detector to the fraction collector approx. 1 min).

15 : Binding curve of the ³ H-propionyl-Lys ¹³ -PTH (1-34) amide obtained according to the Nvoc strategy on the PTH1 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.

16 : HPLC chromatogram (220 nm) of N-terminal Nvoc-protected Lys ⁴ -hPP. (R _t = 24.5 min, purity> 95%)

17 : DAD spectrum of the HPLC chromatogram of N-terminal Nvoc-protected Lys ⁴ -hPP 16 , The lower part shows the DAD spectrum of 200–400 nm, the upper part shows the absorption of the peptide bonds at 220 nm (upper line) and the absorption of the Nvoc group at 350 nm (lower line).

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

Bibliography:

• [1] 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-Verlag.
• [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, JP 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] Gabrele, C., Wieland, H.A., Koglin, N., Stidsen, C. and Beck-Sickinger, A.G., Ala (31) –Alb (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) 1, 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, AT. and Sofas, D., Light-directed, spatially addressable parallel chemical synthesis. Science, (1991) 251, 767-73.
• [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, 776-777
• [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
• [11] Kaiser, E., Colescott, R.L., Bossinger, C.D. and cook, P.I., Color test for detection of free terminal amino groups in the solid phase synthesis of peptides. Anal. Biochem., (1970) 34, 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, I.Z., Reactivity of N-hydroxysuccinimide ester. Pole. J. Chem., (1992) 66, 111-118
• [14] Tang, YS, Davis, AM and Kitcher, JP, N-succinimidyl propionate: characterization and optimum condi tions 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, D.J. and Kling, P.J., Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies 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, F.R., McClure, I., Keutmann, H.T., Potts, J.T., Kronenberg, H.M., Abou-Samra, A.B., Segre, G.V. and Gardella, T.J., The extracellular amino-terminal region of the parathyroid hormone (PTH) / PTH-related peptide receptor determines the binding affinity for carboxyl-terminal fragments of PTH- (1-34). Endocrinology, (1994) 134, 879-884

List of abbreviations

2-Cl-Z-

2-Chlorobenzyloxycarbonyl

ACN

Acetonitril

Boc-

tert.Butoxycarbonyl-

Bzl

Benzyl-

DAD

Dioden-Array-Detektor

DCM

Dichlormethan

Dde-

1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-

DIC

N,N-Di-isopropyl-carbo-di-imid

DIPEA

N,N-Di-isopropyl-ethyl-amin

DMF

N,N-Dimethylformamid

ESI-MS

Elektrospray-Ionisation Massenspektrometrie

Fmoc-

(9-Fluoro-enyl)methoxycarbonyl-

geb

gebunden

³H

Tritium

HF

Fluorwasserstoff Flusssäure

HOBt

1-Hydroxy-benzotriazol

HPLC

High Performance Liquid Chromatography

ivDde-

1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)-3-methylbutyl-

K

Lysin

Bq

Becquerel

m

Masse

Mmt

4-Methoxytrityl-

Mtt

4-Methyltrityl-

NHS

N-Hydroxysuccinimid

N^α

Stickstoff der α-Aminogruppe

N^ε

Stickstoff der ε-Aminogruppe (z. B. in Lysin)

NPY

Neuropeptid Y

Nvoc

Nitroveratryloxycarbonyl

P

Prolin

PP

pankreatisches Polypeptid

PTH

Para-Thyroid-Hormon

S

Serin

SG

Schutzgruppe

T

polymerer Träger

TIS

Triisopropylsilan

tBu

tert.-Butyl

TFA

Trifluoressigsäure (Trifluoro-acetic acid)

Tfa

Trifluoracetyl

UV

Ultraviolett

X

beliebige Aminosäure

Y

Tyrosin

Z-

Benzyloxycarbonyl.

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

2-Cl-Z

2-chlorobenzyloxycarbonyl

ACN

acetonitrile

Boc

tert.butoxycarbonyl

Bzl

benzyl

DAD

Diode array detector

DCM

dichloromethane

Dde

ethyl 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)

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-

give

bound

³ H

tritium

HF

Hydrogen fluoride hydrofluoric acid

HOBt

1-hydroxy-benzotriazole

HPLC

High performance liquid chromatography

ivDde-

1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) -3-methylbutyl

K

lysine

bq

Becquerel

m

Dimensions

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

protecting group

T

polymeric carrier

TIS

triisopropylsilane

Bu

tert-butyl

TFA

Trifluoroacetic acid

Tfa

trifluoroacetyl

UV

Ultraviolet

X

any amino acid

Y

tyrosine

Z-

Benzyloxycarbonyl.

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

Claims (12)

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 building blocks on a carrier material, characterized in that a.) In the peptide synthesis, an amino acid building block is incorporated in at least one position to be labeled , which is an amino group which is protected by a group which differs functionally from the protective groups on the amino groups 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 marked via amino-reactive substances, d.) the protective groups on the amino groups not to be marked are subsequently cleaved off and the selectively marked peptide thus obtained is purified in a known manner. A method according to claim 1, characterized in that at the peptide synthesis, the amino groups not to be labeled, photolabile Wear protective groups. A method according to claim 1, characterized in that at the peptide synthesis, the amino groups not to be labeled, initially hydrazine-labile Protecting groups or protecting groups with a dilute acid, such as 1 vol.% Trifluoroacetic acid (TFA) in dichloromethane (DCM), can be split off. A method according to claim 3, characterized in that the Protective groups of all amino groups not to be labeled subsequently selectively to peptide synthesis and before detaching the peptide from the support material are removed and the non-labeled amino groups with photolabile Protection groups newly protected and then the peptide of the solid carrier material superseded becomes. A method according to claim 1, characterized in that the The amino acid building blocks used in the peptide synthesis do not adhere to the labeling amino groups, base-labile protecting groups, preferably 9-Fluoro-enyl) methoxycarbonyl (Fmoc) groups, wear after installation any amino acid building block that is not to be labeled, the base-labile Protective group is split off and the amino group not to be labeled is newly protected with photolabile protective groups. Method according to one of claims 1 to 5, characterized in that that during the synthesis the amino groups to be labeled are protective groups, preferably tert-butoxycarbonyl (Boc) groups, which do not with diluted acids, like 1 vol.% TFA in DCM, but at high acid concentrations, like over 50 vol.% TFA in DCM, can be split off. Method according to one of claims 2 to 4, characterized in that that the photolabile protecting groups of the ortho-nitrobenzyl type, preferably Nitroveratryloxycarbonyl (Nvoc) groups. A 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- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) -3-methylbutyl- (ivDde) groups. A method according to claim 3, characterized in that the with a diluted one Removable acid Protecting groups trityl compounds, preferably 4-methoxytrityl- (Mmt) or 4-methyltrityl (Mtt) groups. Method according to one of claims 1 to 9, characterized in that that the label is a radioactive isotope. 1'1. method according to one of the claims 1 to 10, characterized in that as amino-reactive substances N-hydroxysuccinimide (NHS) esters can be used. A method according to claim 11, characterized in that a radioactive NHS ester, preferably N-succinimlidyl- [2,3- ³ H] propionate, is used for labeling. Use of peptides in which all amino groups that are not to be labeled are protected by protective groups, preferably photolabile protective groups, and at least one amino group to be labeled unprotected, for the production of a selectively radioactively labeled peptide.