DE10319611A1 - New conjugates useful as reagents for mass-spectrometric analysis of proteins, comprise a protein-reactive group covalently bound to an affinity ligand or solid phase through a linker - Google Patents

Abstract

Conjugates (I) comprising a protein-reactive group covalently bound to an affinity ligand or solid phase through a linker are new. Conjugates of formula (I) comprising a protein-reactive group covalently bound to an affinity ligand or solid phase through a linker are new: [Image] A : an affinity ligand or solid phase; S : B-X-O-Ar-CH2-O; B : O, NH or OC(O) and is bonded to A; X : 1-10C alkylene; Ar : p-phenylene with 0-2 electron-donating substituents; Z : CO-(CH2)x-CHRx-NH; Rx : an amino acid side chain; x : 0-5; L' : a bridging group capable of covalently bonding to two N atoms; Z' : NH-CHRy-(CH2)y-CO in which the orientation of the CO and NH groups is opposite to that in Z; Ryan amino acid side chain; y : 0-5; R, R' : alpha -amino acid side chains; k, l, m, n, o, p, q : 0-10, provided that k+l+m+n+o+p+q = 1-40; and PRG : a protein-reactive group. Independent claims are also included for: (1) a process for preparing (I); and (2) a kit for mass-spectrometric analysis of proteins, comprising one or more differently isotope-labeled compounds (I).

Description

The The invention relates to new, isotope-encoded affinity markers for mass spectrometric analysis of proteins and their production, their use and kits containing them.

The Proteomics technology opened the possibility, by analyzing biological systems at the protein level identify biological targets and markers. It is known, that only one of the proteins encoded in the genome Part of all possible Proteins is expressed, for example tissue type, development status, Activation of receptors or cellular interactions the expression pattern and rates affect. To differences in the expression of proteins Finding in healthy or diseased tissue can be various comparative Methods for analyzing protein expression patterns ((a) S.P. Gygi et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 9390; (b) D. R. Goodlett et al., Proteome Protein Anal., 2000, 3; (c) S.P. Gygi et al., Curr. Opin. Biotechnol. 2000, 11, 396).

A powerful method is the mass spectrometric detection of proteins. When using different isotope-coded affinity markers (ICAT ^® = Isotope Coded Affinity Tags) and tandem mass spectrometry, this method can be used for the quantitative analysis of complex protein mixtures ((a) SP Gygi et al., Nature Biotechnology, 1999, 17, 994; ( b) RH Aebersold et al., WO 00/11208). The method is based on the fact that each of two or more protein mixtures to be compared, which have been obtained in different cell states, is reacted with an affinity marker of a different isotope coding. The protein mixtures are then combined, optionally fractionated or proteolytically treated and purified by affinity chromatography. After elution of the bound fragments, the eluates are analyzed by combining liquid chromatography and mass spectrometry (LC-MS). Pairs or groups of peptides labeled with affinity markers that differ only in the isotope coding are chemically identical and are eluted almost simultaneously in HPLC, but they differ in the mass spectrometer by the respective molecular weight differences due to different isotope patterns of the affinity markers. Relative protein concentrations can be obtained directly by measuring the peak areas. Suitable affinity markers are conjugates of affinity ligands which are covalently linked to protein-reactive groups via bridge members. Different isotopes are built into the bridge members. The method was described using affinity markers in which hydrogen atoms were replaced by deuterium atoms ( ¹ H / ² D isotope coding).

The method described in the prior art with ¹ H / ² D-isotope-coded affinity markers has various disadvantages, in particular an isotope effect of the differently labeled but otherwise identical peptide fragments in the LC, inadequate stability of the affinity markers in general and especially in LC-MS / MS, a lack of efficiency in avidin monomer-based affinity chromatography, a lack of flexibility in incorporating the isotope labels.

task The present invention was to provide improved isotope-encoded Affinity tag.

haben, in der
A der Affinitätsliganden-Rest oder die feste Phase ist,
S eine Gruppe der Formel

ist, in der
B O, NH oder OC(=O) und an A gebunden ist,
X eine lineare oder verzweigte Gruppe der Formel (CH₂)_a ist,
worin a eine Zahl von 1 bis 10 ist,
Y eine elektronenschiebende Gruppe ist und
b die Zahl 0, 1 oder 2 ist,
Z ein jeweils unabhängig von anderen Z der gleichen oder einer anderen Laufvariable ausgewählter Aminosäure-Rest CO-(CH₂)_x-CHR^x-NH mit der Aminosäure-Seitenkette R^x und der Zahl x aus dem Wertebereich von 0 bis 5 ist,
L' eine jeweils unabhängig von anderen L' der gleichen oder der anderen der beiden Laufvariablen lund m ausgewählte Brücke ist, die eine kovalente Verknüpfung zweier Stickstoffatome ermöglicht oder erleichtert,
Z' ein jeweils unabhängig von anderen Z' der gleichen oder einer anderen Laufvariable ausgewählter Aminosäure-Rest NH-CHR^y-(CH₂)_y-CO mit der Aminosäure-Seitenkette R^y und der Zahl y aus dem Wertebereich von 0 bis 5 ist, der sich von Z in der unterschiedlichen Orientierung bezüglich der terminalen CO- und NH-Gruppe unterscheidet,
R und R' an einem Piperazin-Ring unabhängig voneinander und unabhängig von anderen R und R' an anderen Piperazin-Ringen der gleichen oder der anderen der beiden Laufvariablen 1 und m jeweils eine α-Aminosäure-Seitenkette ist,
k, l, m, n, o, p, q unabhängig voneinander jeweils für eine Zahl von 0 bis 10 stehen, wobei die Summe k + l + m + n + o + p + q mindestens 1 und höchstens 40 beträgt, und
PRG die proteinreaktive Gruppe ist,
oder deren Salze.The invention relates to compounds having a protein-reactive group and an affinity ligand residue or a solid phase and a linker covalently linking them, which have the formula

have in the
A is the affinity ligand residue or the solid phase,
S is a group of the formula

is in the
BO, NH or OC (= O) and is bound to A,
X is a linear or branched group of the formula (CH ₂ ) _a ,
where a is a number from 1 to 10,
Y is an electron-donating group and
b is the number 0, 1 or 2,
Z is an amino acid residue CO- (CH ₂ ) _x -CHR ^x -NH with the amino acid side chain R ^x and the number x from the value range from 0 to 5, which is selected independently of other Z of the same or a different run variable.
L 'is a bridge selected independently of other L' of the same or the other of the two run variables l and m, which bridge enables or facilitates a covalent linkage of two nitrogen atoms,
Z 'is an amino acid residue NH-CHR ^y - (CH ₂ ) _y -CO with the amino acid side chain R ^y and the number y from the value range from 0 to 5, which is selected independently of other Z' of the same or a different run variable , which differs from Z in the different orientation with regard to the terminal CO and NH groups,
R and R 'on a piperazine ring independently of one another and independently of other R and R' on other piperazine rings of the same or the other of the two run variables 1 and m are each an α-amino acid side chain,
k, l, m, n, o, p, q each independently represent a number from 0 to 10, the sum k + l + m + n + o + p + q being at least 1 and at most 40, and
PRG is the protein reactive group
or their salts.

The compounds of the invention are suitable as affinity markers for mass spectrometric analysis of proteins and are below also as an affinity marker designated. You point towards that State of the art in particular the following advantages.

Deuterium-labeled, ^13C-labeled or ¹³ C and ¹⁵ N-labeled glycine-blocks or other appropriately labeled amino acid building blocks are inexpensive starting materials, which allow a flexible design of the isotopically labeled affinity tag. In the affinity marker described in the formula (I), more than twenty ¹³ C labels and additionally more than ten ¹⁵ N isotopes can be introduced in a simple manner. In contrast to the previously described affinity markers with a significantly smaller mass difference (ΔM = 8), the parallel analysis of several proteome samples is also possible by modification with affinity markers with different mass differences. The version with up to 4 differently labeled affinity markers is preferred, which enables the simultaneous analysis and relative quantification of up to 4 complex proteome samples.

The modular structure of the affinity markers allows a flexible, adapted to the requirements of the workflow Combination of the individual building blocks.

at the group S of the affinity markers according to the invention it is an acid labile Predetermined breaking point (acid-cleavable Group), which decomplexes the peptide fragments by a much more efficient, for example on streptavidin or oligomeric Avidin based affinity chromatography for the Biotin-modified peptide fragments or through a reversible binding to a fixed phase. Farther have after acid splitting parts of the affinity markers remaining on the peptide fragments, so-called Tags, low molecular weight and high isotope density.

The The predetermined breaking point S is acid-labile functionality the under the influence of acid a split in the affinity marker guaranteed for example facilitate the release of the affinity column from the peptide remaining remaining to downsize or overall work processes to make it more efficient. The acid lability of S can be modulated with b electron-shifting groups Y. The group B enables or facilitates the binding of S to the affinity ligand, by way of example and preferably by O or NH, or to the solid Phase, for example and preferably by the oxycarbonyl group OC (= O).

The Handling the affinity markers is improved solubility, by a crystalline or amorphous appearance as well as by increased stability improved.

In the context of the present invention, the following terms, substituents and symbols, unless expressly stated otherwise, have the following meaning:
Alkyl per se and "alk" and "alkyl" in alkylene, alkenylene, alkynylene, alkoxy and alkylamino stand for a linear or branched alkyl radical with generally 1 to 6, preferably 1 to 4, particularly preferably 1 to 3 carbon atoms, for example and preferably for methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.

alkoxy is exemplary and preferably methoxy, ethoxy, n-propoxy, Isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

alkylamino stands for an alkylamino radical with one or two (independently selected) alkyl substituents, exemplary and preferably for Methylamino, ethylamino, n-propylamino, isopropylamino, tert-butylamino, n-pentylamino, n-hexylamino, N, N-dimethylamino, N, N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino, N-t-butyl-N-methylamino, N-ethyl-N-n-pentylamino and N-n-hexyl-N-methylamino.

alkylene, Alkenyls and alkynes stand for divalent alkyl groups in the case of alkenylene or alkynylene one, two, three, four, five or more double or triple bonds and accordingly at least Have 2 carbon atoms, for example and preferably for methylene, Ethylene, ethenylene, ethynylene, n-propylene, iso-propylene, n-propenylene, Methylethenylene and propinylene.

Amino acids and amino acid residues have the formula H ₂ N-CHR ^AS - (CH ₂ ) _as -COOH or NH-CHR ^AS - (CH ₂ ) _as -CO with the amino acid side chain R ^AS and the number as from the value range of 0 to 5, by way of example and preferably α-amino acids (as = 0), in particular the 20 natural proteinogenic L-α-amino acids, particularly preferably glycine, leucine and alanine, and ω-amino acids (as = 1, 2, 3, 4 or 5), by way of example and preferably H ₂ N- (CH ₂ ) ₂ -COOH, or their amino acid residues. The amino acids and amino acid residues are optionally in the D, L or racemic form.

arylene stands for a bivalent mono- to tricyclic aromatic, carbocyclic Radical usually having 6 to 14 carbon atoms; example, and preferably for Phenylene, naphthylene and phenanthrenylene.

Electron-donating Group stands for one a substitute with a positive inductive (+ I-) and / or positive mesomeric (+ M-) effect, exemplary and preferably for alkoxy or alkylamino.

halogen stands for Fluorine, chlorine, bromine and iodine.

The affinity ligand A is used for the selective enrichment of samples using affinity chromatography. The pillars of affinity are provided with the corresponding complementary partners to the affinity ligands, which are covalent or non-covalent bonds with the affinity ligands received. An example for a suitable affinity ligand is biotin or a biotin derivative that works with the complementary peptides avidin or streptavidin forms strong, non-covalent bonds. On this way Specimens to be examined selectively using affinity chromatography be isolated from sample mixtures. In the same sense, for example also carbohydrate residues that do not have covalent interactions For example, fixed lectins can enter as an affinity ligand be used. The interaction can continue in the same sense of haptens with antibodies or the interaction of transition metals with appropriate ligands can be used as complexing agents or other systems that interact with each other.

Alternatively, the targeted depletion can also be achieved by selective, reversible connection to an appropriately functionalized solid phase A. Suitable solid phases are, for example, amino-functionalized resins based on silica gel and are also known from the peptide syntheses carried out as solid-phase syntheses, such as trityl resin, sasrin resin based on benzyl alcohol support, Wang resin based on benzyl alcohol support, Wang polystyrene resin, Rink amide MBHA resin or TCP (trityl chloride polystyrene) resin (in the formulas shown, the circled P stands for the rest of the resin):

Prefers is as solid phase A a polymeric carrier, especially a modified one natural or synthetic resin, for example a resin on silica gel or polyethylene glycol base, with suitable for connecting group S. functional groups, for example hydroxyl, carboxy and amino groups, especially hydroxy groups. Is particularly preferred as the solid phase A is a functionalized silica gel-based resin, for example an aminopropyl silica gel, e.g. from Aldrich under number 36425-8 is distributed.

Reactive protein PRG groups are used for the targeted labeling of proteins on selected functional ones Groups. PRGs have a specific reactivity for terminal functional groups of the proteins. amino acids as elements of proteins, often used for targeted labeling are, for example, mercaptoaminomonocarboxylic acids such as Cysteine, diaminomonocarboxylic acids such as lysine or arginine or monoaminodicarboxylic acids such as aspartic acid or Glutamic acid. Can continue protein-reactive groups such as phosphate-reactive groups such as Metal chelates and aldehyde-reactive and ketone-reactive groups such as for example amines with subsequent treatment with sodium borohydride or be sodium cyanoborohydride. It can also be groups that follow a targeted protein derivatization such as one Bromocyanine cleavage or elimination of phosphate groups etc. react with the implementation products.

suitable Accordingly, protein-reactive groups PRG show examples and preferably selective reactivity for a terminal functionality of a Amino acid, for one Phosphate group or for one possibly previously generated aldehyde or keto group in the protein or for the reaction product a targeted implementation of the protein.

suitable protein reactive groups PRG have also been described, for example, by W.H. Scouting in Methods in Enzymology, Volume 135, edited by Klaus Mosbach, AcademicPress Inc. 1987, p. 30ff.

In a special embodiment of the invention, the protein-reactive group PRG has solubility-improving functional groups.

Preferred compounds of the formula (I) according to the invention are those in which
A is the affinity ligand residue or the solid phase,
in the formula of group S
BO, NH or OC (= O),
X is a linear group of the formula [CH ₂ ] _a ,
where a is a number from 1 to 10,
Y is alkoxy or dialkylamine and
b is the number 0, 1 or 2,
Z is the amino acid residue of glycine, leucine or alanine, independently of other Z of the same or a different run variable,
L 'is a bridge selected independently of other L' of the same or the other of the two run variables 1 and m, which is composed of building blocks selected from alkylene, alkenylene, alkynylene, arylene, CO, CS and NH, the terminal building blocks being composed of CO and CS are selected,
Z 'is the amino acid residue of glycine, leucine or alanine, independently of other Z' of the same or a different run variable,
R and R 'on a piperazine ring are each the same amino acid side chain,
PRG is the protein reactive group
and the sum k + l + m + n + o + p + q is at least 1 and at most 20.

-CO-[CH₂]_r-I mit r = 1, 2, 3, ..., 10,
-CO-[CH₂]_r-Br mit r = 1, 2, 3, ..., 10,
-CO-[CH₂]_r-Cl mit r = 1, 2, 3, ..., 10 und
-CO-CH=CH₂,
und die Summe k + l + m + n + o + p + q mindestens 1 und höchstens 10 beträgt.Preferred compounds of the formula (I) according to the invention are those in which
A is the rest of biotin or a biotin derivative,
in the formula of group S
Is BO or NH,
X is a linear group of the formula [CH ₂ ] _a ,
where a is the number 1, 2 or 3,
Y is alkoxy or dialkylamine and
b is the number 0 or 1,
Z is the amino acid residue of glycine, leucine or alanine, independently of other Z of another run variable,
L 'is a bridge selected independently of other L' of the same or the other of the two run variables l and m, which is composed of 2 to 10 units selected from alkylene, alkenylene, alkynylene, arylene, CO, CS and NH, the are terminal building blocks made of CO,
Z 'is the amino acid residue of glycine, leucine or alanine, independently of other Z' of another run variable,
R and R 'are each hydrogen,
-PRG is selected from

-CO- [CH ₂ ] _r -I with r = 1, 2, 3, ..., 10,
-CO- [CH ₂ ] _r -Br with r = 1, 2, 3, ..., 10,
-CO- [CH ₂ ] _r -Cl with r = 1, 2, 3, ..., 10 and
-CO-CH = CH ₂ ,
and the sum k + l + m + n + o + p + q is at least 1 and at most 10.

preferred are such compounds according to the invention of formula (I) in which all R and R 'are hydrogen.

Those compounds of the formula (I) in which A is preferred for the acyl radical of an affinity ligand, by way of example and preferably biotinyl or a biotin derivative, or for one with a polymeric carrier, are also preferred ger connected functional group, for example and preferably a carrier-bound hydroxy, carboxy or amino group, in particular a carrier-bound amino group.

Prefers are also those compounds of formula (I) in which PRG a electrophilic group, for example and preferably an epoxy, a Maleimido group, a halogen or a vinyl group. Preferred compounds of the formula (I) are also those in which PRG a suitable bridge, which enables the electrophilic group to be attached to Z or relieved. Furthermore, such a group can be replaced by appropriate execution additionally the solubility improve.

-CO-[CH₂]_r-I mit r = 1, 2, 3,..., 10,
-CO-[CH₂]_r-Br mit r = 1, 2, 3,..., 10,
-CO-[CH₂]_r-Cl mit r = 1, 2, 3,..., 10 und
-CO-CH=CH₂. Preference is also given to those compounds of the formula (I) in which PRG has an electrophilic group and a suitable bridge. Among these, particular preference is given to those compounds of the formula (I) in which -PRG is selected from the bridged electrophiles

-CO- [CH ₂ ] _r -I with r = 1, 2, 3, ..., 10,
-CO- [CH ₂ ] _r -Br with r = 1, 2, 3, ..., 10,
-CO- [CH ₂ ] _r -Cl with r = 1, 2, 3, ..., 10 and
-CO-CH = CH ₂ .

Prefers are also those compounds of formula (I) in which B in the Formula of group S for O or NH stands. Such compounds are particularly preferred of formula (I) in which B in the formula of group S represents NH.

Preference is also given to those compounds of the formula (I) in which X in the formula of the group S is a linear group of the formula [CH ₂ ] _a , in which a is a number from 1 to 10, by way of example and preferably 1, 2 or 3, is.

Prefers are also those compounds of formula (I) in which Y in the Formula of the group S alkoxy or dialkylamine, by way of example and preferably Methoxy, is.

Prefers are also those compounds of formula (I) in which b in the Formula of group S is the number 0 or 1.

Z and Z 'are the same or different amino acid residues.

Preferred compounds of the formula (I) are those in which Z is the rest of glycine, leucine or is alanine. Z is not isotope-labeled or isotope-labeled, for example and preferably with ¹³ C or ¹⁵ N isotopes or one. Combination of these isotopes.

Preferred compounds of the formula (I) are also those in which Z 'is the residue of glycine, leucine or alanine. Z is not isotope-labeled or isotope-labeled, for example and preferably with ¹³ C or ¹⁵ N isotopes or a combination of these isotopes.

Prefers are particularly those compounds of formula (I) in which Z and Z 'independently of each other selected are from the amino acid residues of glycine, leucine and alanine.

Also preferred are compounds of formula (I) in which Z is the glycine residue CO-CH ₂ -NH or Z 'is the glycine residue NH-CH ₂ -CO. Among these, particular preference is given to those compounds of the formula (I) in which Z is the glycine residue CO-CH ₂ -NH and Z 'is the glycine residue NH-CH ₂ -CO.

Prefers are also those compounds of the formula (I) in which L 'consists of the building blocks Alkylene, especially linear alkylene, alkenylene, alkynylene, arylene, CO, CS and NH, for example and preferably from a number of 2 to 10 such building blocks is constructed, the terminal Blocks selected from CO and CS, especially CO, are.

Of these, particular preference is given to those compounds of the formula (I) in which L 'is selected from CO-CO, CO- (CH ₂ ) _s -CO, CO-arylene-CO, CO-CH ₂ -NH-CO-NH- CH ₂ -CO, CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO, CO-CH ₂ -NH-CO-CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO- (CH ₂ ) _s -CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO-arylene-CO-NH-CH ₂ -CO, CO and CS, where s is the number 1, 2, 3, 4 , 5 or 6.

In a particular embodiment, L 'contains one or more further amino acid residues such as Z and Z', in particular glycine residues, which can contain ¹³ C or ¹⁵ N isotope labels or a combination of these labels.

Preference is also given to those compounds of the formula (I) in which R and R 'on a piperazine ring are each the same amino acid side chain, in particular hydrogen. Among these, particular preference is given to those compounds of the formula (I) in which R and R 'on all piperazine rings of one of the two run variables n and p are the same amino acid side chains, in particular hydrogen. Among these, particular preference is given to those compounds of the formula (I) in which R and R 'on all piperazine rings are the same amino acid side chains, in particular hydrogen. The piperazine rings are present by way of example and are preferably unlabeled or with ¹² C or ¹⁵ N labels or with a combination of these labels.

Prefers are also those compounds of formula (I) in which the sum k + l + m + n + o + p + q at most 20, especially at most 10.

A third function of amino acids which may be present in an amino acid side chain R, R ', R ^x or R ^y can optionally be present freely or protected with a protective group. In a special embodiment, the side chains contribute to the improved solubility of the affinity markers.

The non-isotope-labeled compounds already represent isotope coding. For further isotope coding, the compounds according to the invention are labeled by the targeted incorporation of isotopes. In a corresponding embodiment, the compounds according to the invention are labeled with one or more, the same or different isotopes. The isotopes are exemplary and preferably selected from the group comprising ² D, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O and ³⁴ S.

In a preferred isotope-labeled embodiment, the compounds according to the invention are isotope-labeled with at least one carbon atom of the isotope ¹³ C, in particular four to 20 ¹³ C atoms. The disadvantageous isotope effect in the LC observed with ¹ H / ² D isotope-coded affinity markers is significantly reduced or even not at all pronounced with ¹² C / ¹³ C isotope coding. As an alternative or in addition to this ¹³ C label, the isotopes ² D, ¹⁵ N, ¹⁷ O, ¹⁸ O and / or ³⁴ S can also be used.

In a particular embodiment of the invention, ¹³ C-labeled compounds are additionally isotopically labeled with at least one nitrogen atom of the ¹⁵ N isotope, preferably one to ten ¹⁵ N atoms, in particular one to four ¹⁵ N atoms.

The isotope labels are usually in Z, Z ', L', the piperazine rings, R, R 'and / or PRG made, for example and preferably in Z, Z ', L' and / or the piperazine rings.

to Improve solubility the affinity marker according to the invention can existing acidic and / or basic functional groups in the form their salts, preferably their hydrochlorides, acetates, trifluoroacetates, Alkali metal salts or ammonium salts, produced and used become.

The in the respective combinations or preferred combinations of Residues specified in detail are independent of the respective specified combinations of the residues as desired replaced by residual definitions of another combination.

All combinations of two or more of the are particularly preferred preferred embodiments mentioned above.

• i) ein Affinitätsligand A-OH oder A-NH₂ oder eine Hydroxy-, Carboxy- oder Amino-funktionalisierte feste Phase A-OH, A-COOH oder A-NH₂ oder eine aktivierte Form davon mit einer Verbindung der Formel
  
  in der B, X, Y und b die gleiche Bedeutung wie in Formel (I) haben, oder deren Derivat, in dem die benzylische Hydroxygruppe eine Schutzgruppe trägt, zu einer Verbindung der Formel
  
  in der A, B, X, Y und b die gleiche Bedeutung wie in Formel (II) haben, oder deren Derivat mit geschützter Hydroxygruppe umgesetzt wird,
• ii) a) ein geschütztes Intermediat der Formel
  
  in der SG und SG' für zwei orthogonale Schutzgruppen stehen und Z, L', Z', R, R', k, l, m, n, o, p und q die gleiche Bedeutung wie in Formel (I) haben, hergestellt wird, aus dem Intermediat der Formel (IV) die Schutzgruppe SG abgespalten wird und die Verbindung der Formel (III) oder deren geschütztes Derivat, wobei im Falle des Derivats zunächst noch die Schutzgruppe abgespalten wird, mit dem entschützten Intermediat der Formel (IV) zu einer Verbindung der Formel
  
  in der A, S, Z, L', Z', R, R', k, l, m, n, o, p, q und SG' die gleiche Bedeutung wie in den Formeln (I), (III) und (IV) haben, gekuppelt wird, oder b) die Verbindung der Formel (III) oder deren geschütztes Derivat, wobei im Falle des Derivats zunächst noch die Schutzgruppe abgespalten wird, mit einer Verbindung der Formel HO-(Z)_k-(Z)_q-SG' (VI),in der Z die gleiche Bedeutung wie in Formel (I) hat, k und q unabhängig voneinander jeweils für eine Zahl von 0 bis 10 stehen, wobei die Summe k + q mindestens 1 beträgt, und SG' für eine Schutzgruppe steht, zu einer Verbindung der Formel A-S-(Z)_k-(Z)_q-SG' (VII),in der A und S die gleiche Bedeutung wie in Formel (III) haben und Z, k, q und SG' die gleiche Bedeutung wie in Formel (VI) haben, gekuppelt wird, oder c) die Verbindung der Formel (III) oder deren geschütztes Derivat, wobei im Falle des Derivats zunächst noch die Schutzgruppe abgespalten wird, mit einer Verbindung der Formel HO-(Z)_k-SG' (VI'),in der Z die gleiche Bedeutung wie in Formel (I) hat, k für eine Zahl von 1 bis 10 steht und SG' für eine Schutzgruppe steht, zu einer Verbindung der Formel A-S-(Z)_k-SG' (VII'),in der A, S und Z die gleiche Bedeutung wie in Formel (I) haben, k für eine Zahl von 1 bis 10 steht und SG' für eine Schutzgruppe steht, gekuppelt wird, ein einseitig geschütztes Intermediat der Formel
  
  in der V für HO oder H₂N steht, Z, L', Z', R, R' die gleiche Bedeutung wie in Formel (I) haben, l, m, n, o, p, q unabhängig voneinander jeweils für eine Zahl von 0 bis 10 stehen, wobei die Summe l + m + n + o + p + q mindestens 1 beträgt und höchstens den Wert der Differenz 40 – k hat, wobei k die Zahl von 1 bis 10 gemäß Formel (VII') ist, und SG' für eine Schutzgruppe steht, hergestellt wird, aus der Verbindung der Formel (VII') die Schutzgruppe SG' abgespalten wird und die Verbindung der Formel (VIII) mit der entschützten Verbindung der Formel (VII') zu einer Verbindung der Formel (V), wobei k gleich oder größer als l ist, gekuppelt wird, und
• iii) aus der in Schritt ii) nach Variante a) erhaltenen Verbindung der Formel (V) oder b) erhaltenen Verbindung der Formel (VII) oder c) erhaltenen Verbindung der Formel (V), wobei k gleich oder größer als 1 ist, die Schutzgruppe SG' abgespalten wird und
• iv) die in Schritt iii) erhaltene entschützte Verbindung schließlich mit dem Derivat einer proteinreaktiven Gruppe oder der aktivierten Vorstufe des Derivats einer proteinreaktiven Gruppe der Formel U-PRG (IX),in der U für eine Gruppe steht, die die Verknüpfung von PRG mit dem entsprechenden Stickstoffatom von Z oder einem Piperazin-Ring ermöglicht, und PRG die gleiche Bedeutung wie in Formel (I) hat, umgesetzt wird.

The invention further relates to a method for producing a compound according to the invention, in which

• i) an affinity ligand A-OH or A-NH ₂ or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH, A-COOH or A-NH ₂ or an activated form thereof with a compound of the formula
  
  in which B, X, Y and b have the same meaning as in formula (I), or their derivative in which the benzylic hydroxyl group bears a protective group, to give a compound of the formula
  
  in which A, B, X, Y and b have the same meaning as in formula (II), or their derivative is reacted with a protected hydroxy group,
• ii) a) a protected intermediate of the formula
  
  in which SG and SG 'represent two orthogonal protective groups and Z, L', Z ', R, R', k, l, m, n, o, p and q have the same meaning as in formula (I) the protective group SG is split off from the intermediate of the formula (IV) and the compound of the formula (III) or its protected derivative, with the protective group being split off first in the case of the derivative, with the deprotected intermediate of the formula (IV) a compound of the formula
  
  in the A, S, Z, L ', Z', R, R ', k, l, m, n, o, p, q and SG' the same meaning as in the formulas (I), (III) and (IV), is coupled, or b) the compound of the formula (III) or its protected derivative, in the case of the derivative the protective group being split off first with a compound of the formula HO- (Z) _k - (Z) _q -SG '(VI), in which Z has the same meaning as in formula (I), k and q each independently represent a number from 0 to 10, the sum k + q being at least 1, and SG 'representing a protective group, to form a compound of the formula AS- (Z) _k - (Z) _q -SG '(VII), in which A and S have the same meaning as in formula (III) and Z, k, q and SG 'have the same meaning as in formula (VI) is coupled, or c) the compound of formula (III) or its protected derivative, in which case the protective group is first split off with a compound of the formula HO- (Z) _k -SG '(VI'), in which Z has the same meaning as in formula (I), k stands for a number from 1 to 10 and SG 'stands for a protective group, to a compound of the formula AS- (Z) _k -SG '(VII'), in which A, S and Z have the same meaning as in formula (I), k stands for a number from 1 to 10 and SG 'stands for a protective group, is coupled, an unilaterally protected intermediate of the formula
  
  in which V represents HO or H ₂ N, Z, L ', Z', R, R 'have the same meaning as in formula (I), l, m, n, o, p, q each independently of one another Are from 0 to 10, the sum l + m + n + o + p + q being at least 1 and at most having the value of the difference 40 - k, where k is the number from 1 to 10 according to formula (VII ') , and SG 'stands for a protective group, is prepared, the protective group SG' is split off from the compound of the formula (VII ') and the compound of the formula (VIII) with the deprotected compound of the formula (VII') to a compound of the formula (V), where k is equal to or greater than l, is coupled, and
• iii) from the compound of formula (V) or b) obtained in step ii) according to variant a) compound of formula (VII) or c) obtained compound of formula (V), wherein k is equal to or greater than 1, the Protection group SG 'is split off and
• iv) the deprotected compound obtained in step iii) finally with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula U-PRG (IX), in which U represents a group which enables the linking of PRG to the corresponding nitrogen atom of Z or a piperazine ring, and PRG has the same meaning as in formula (I).

The Affinity ligands used or produced in the process as starting materials or functionalized solid phases and compounds of the formulas (II), (IV), (VI), (VI '), (VIII) and (IX) are known or can be carried out analogously to known processes unless otherwise stated. The individual reactions are carried out under normal conditions, as far as nothing else is described below.

The Reaction to the compound of formula (III) in step i) of the process usually takes place under the suitable coupling conditions. An activated form of the affinity ligand or the functionalized solid phase is exemplary and preferred an activated ester or an acid chloride.

The Preparation of the intermediate of formula (IV) in variant a) of Step ii) of the process and the intermediate of the formula (VIII) in variant c) of step ii) of the process follows classical methods of peptide chemistry, as known to the person skilled in the art and how they are, for example, in Houben Weyl; Methods of organic Chemistry; Fourth edition; Volume XV Parts 1 and 2; Georg Thieme publishing house Stuttgart 1974 or in Hans-Dieter Jakubke and Hans Jeschkeit: amino acids, peptides, proteins; Verlag Chemie, Weinheim 1982 are described.

protecting groups are orthogonal if each selectively split off in the presence of the other can be. A suitable protective group SG is exemplary and preferably a tertiary Butyloxy group (tBu-O). A suitable protective group SG 'is exemplary and preferably a fluorenyl-9-methoxycarbonyl group (Fmoc). SG tBu-O is particularly preferred in compounds of the formula (IV) and SG 'Fmoc. The protection groups SG 'of the connections of the formulas (VI ') and (VIII) can be the same or different and are particularly preferably both Fmoc. Fmoc as a protecting group SG ' exemplarily and preferably cleaved with piperidine. Fmoc as Protection group SG ' exemplary and preferably split off in DMF. Fmoc as a protecting group SG 'becomes an example and particularly preferably cleaved with piperidine in DMF.

The Group U of the compound of formula (IX) enables the linking of PRG with the corresponding nitrogen atom from Z or a piperazine ring in step iv) of the method by way of example and preferably by it becomes a leaving group. Corresponding groups U are exemplary and preferably activated esters, especially N-hydroxysuccinimide esters or chlorides, or groups from which during the coupling in step iv) a leaving group is generated.

The Coupling the deprotected Intermediates of formula (IV) with the compound of formula (III) or deprotect them Derivative in variant a) of step ii) of the method and the coupling the intermediate of formula (VIII) with the deprotected compound of the formula (VII ') in variant c) of step ii) of the method again take place using the classic methods of peptide chemistry.

In all process steps as required the protective groups mentioned are further reversibly removable Protecting groups, as are common in peptide chemistry, can be used. Suitable protective groups are exemplary and preferably those with trifluoroacetic removable tBu-O protective group and tert-butoxycarbonyl protective group (Boc) or the Fmoc protective group which can be split off with piperidine or morpholine. Other suitable protecting groups and the corresponding methods for the introduction and separation are described for example in Jakubke / Jeschkeit: Amino acids, Peptides, proteins; Verlag Chemie 1982 or in Houben-Weyl, methods of organic chemistry, Georg Thieme Verlag Stuttgart, fourth edition, volume 15.1 and 15.2, edited by E. Wünsch.

The individual reactions of the process can occur at different pressure and temperature conditions carried out be, for example at 0.5 to 2 bar and preferably below Normal pressure, or –30 to + 100 ° C and preferably -10 up to + 80 ° C, in suitable solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, Chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned carried out become. In a preferred embodiment of the method one or more reactions in DMF, THF, dichloromethane, a THF / dichloromethane mixture or a dioxane / water mixture at room temperature or under ice cooling and Normal pressure carried out.

The compounds according to the invention can therefore be prepared by various process variants. The block-by-block principle that is reflected in the process steps and variants enables a multitude of conceivable combinations and, with suitable selection of isotope-marked blocks, almost any type, number and placement of the isotope markings can be realized. In compounds according to the invention of the same constitution and configuration, almost any number, preferably up to 30, of isotope labels can be achieved by incorporating ¹³ C and ¹⁵ N isotopes. This opens the way for simultaneous analysis of several, preferably up to 4, complex protein samples.

object the use of one or more is further different from the invention isotope-labeled compounds according to the invention as a reagent for the mass spectrometric analysis of proteins, in particular for the identification of one or more proteins or protein functions in one or more protein-containing samples and for determination the relative expression levels of one or more proteins in one or more protein-containing samples.

object the invention is also a kit for mass spectrometric Analysis of proteins containing one or more as reagents differently isotope-labeled compounds according to the invention,

Examples

It became 7 affinity markers of formula (Ia) in which A is the acyl residue of biotin and are summarized in the table below.

Used abbreviations:

• Boc - tert-butoxycarbonyl
• tBu-O - tert-butyloxy
• DCM - dichloromethane
• DIEA - diisopropylethylamine (Hünig Base)
• DMAP - dimethylaminopyridine
• DMF - dimethylformamide
• DMSO - dimethyl sulfoxide
• EI - electron impact ionization
• ESI - electrospray ionization
• FD - Field desorption
• Fmoc - fluorenyl-9-methoxycarbonyl
• h - hour (s)
• HPLC - high-performance liquid chromatography
• MALDI - Matrix-assisted laser desorption ionization
• min - minute (s)
• MS mass spectrometry
• MTBE - methyl tert-butyl ether
• RP - Reverse phase
• RT - room temperature
• TCEP - triscarboxyethylphosphine
• TFA - trifluoroacetic acid
• THF - tetrahydrofuran
• TLC - Thin layer chromatography
• (v / v) - Concentration information in volume per volume
• (w / v) - concentration information in mass per volume

The details of the composition of solvent and eluent mixtures are given as far as not expressly stated otherwise, by specifying the components separately from "/" and reproducing the relative parts by volume. For example, "acetonitrile / water 10/1" means a mixture of acetonitrile and water in a volume ratio of 10 to 1.

Preferred eluents (reference by specifying ¹⁾ etc.):

• ¹⁾ Acetonitrile / water 10/1
• ²⁾ Acetonitrile / water 20/1
• ³⁾ dichloromethane / methanol 99/1
• ⁴⁾ dichloromethane / methanol 97.5 / 2.5
• ⁵⁾ dichloromethane / methanol 20/1
• ⁶⁾ dichloromethane / methanol 10/1
• ⁷⁾ Acetonitrile / water / glacial acetic acid 10/1 / 0.1
• ⁸⁾ Acetonitrile / water / glacial acetic acid 5/1 / 0.2
• ⁹⁾ Acetonitrile / water / glacial acetic acid 10/3 / 1.5
• ¹⁰⁾ dichloromethane / methanol / aq. Ammonia (17%) 15/2 / 0.2
• ¹¹⁾ dichloromethane / methanol / aq. Ammonia (17%) 15/4 / 0.5
• ¹²⁾ Acetonitrile / water / glacial acetic acid 10/5/3

to Production of the exemplary affinity markers described below were initially the biotin derivatives of the educt series 1 and the linker derivatives of Educt series 2 manufactured.

Series 1: Derivatives for Production of predetermined breaking points

General formula (a = 1, 2, 3, ..., 10; b = 0, 1, 2):

General rule:

(b = 1 oder 2) und 28,3 g (204,7 mmol) Kaliumcarbonat wurden in 200 ml Aceton zusammengegeben. Es wurde über Nacht bei 60°C gerührt. Anschließend wurde auf Raumtemperatur gekühlt, das Kaliumcarbonat abfiltriert und mit Aceton gewaschen. Die flüssige Phase wurde eingeengt und der Rückstand durch Flash-Chromatographie an Kieselgel (Elutionsgemisch: Dichlormethan/Methanol: 99/1) gereinigt. Die entsprechenden Fraktionen wurden vereinigt und das Lösungsmittel in vacuo abgedampft.13.17 g (49.13 mmol) of N- (3-bromopropyl) phthalimide, 40.94 mmol of the desired hydroxybenzaldehyde of the formula

(b = 1 or 2) and 28.3 g (204.7 mmol) of potassium carbonate were combined in 200 ml of acetone. The mixture was stirred at 60 ° C. overnight. The mixture was then cooled to room temperature, the potassium carbonate was filtered off and washed with acetone. The liquid phase was concentrated and the residue was purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 99/1). The appropriate fractions were combined and the solvent was evaporated in vacuo.

22.63 mmol of this intermediate were in 300 ml dichloromethane / methanol 1/1 solved and cooled to -5 ° C to 0 ° C. 430 mg (11.31 mmol) sodium borohydride was added and the mixture stirred at this temperature. After 20 minutes, a few drops of glacial acetic acid were added until none Gas evolution gave more. Then it was concentrated. The residue was redistilled three times with methanol and then by flash chromatography purified on silica gel (elution mixture: dichloromethane / methanol: 20/1). The appropriate fractions were combined and the solvent evaporated in vacuo.

[ESI-MS: m/e = 182 (M+H)⁺]. S.1.2(a = 3, b = 1)

[ESI-MS: m/e = 212 (M+H)⁺]. S.1.3 (a = 3, b = 1)

[ESI-MS: m/e = 212 (M+H)⁺].19.75 mmol of this intermediate and 4.79 ml (98.77 mmol) of hydrazine monohydrate were combined in 420 ml of ethanol and stirred for one day at room temperature. The precipitated solid became filtered off and washed with ethanol. The mixture was then concentrated and the residue was purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol / aqueous ammonia (17%): 15/4 / 0.5). The appropriate fractions were combined and the solvent was evaporated in vacuo. S.1.1 (a = 3, b = 0)

[ESI-MS: m / e = 182 (M + H) ⁺ ]. S.1.2 (a = 3, b = 1)

[ESI-MS: m / e = 212 (M + H) ⁺ ]. S.1.3 (a = 3, b = 1)

[ESI-MS: m / e = 212 (M + H) ⁺ ].

Educt series 1: biotin derivatives

General formula:

General rule:

[ESI-MS: m/e = 390 (M-OH^–)⁺]. E.1.2

[ESI-MS: m/e = 420 (M-OH^–)⁺]. E.1.3

[ESI-MS: m/e = 420 (M-OH^–)⁺].1.24 g (5.07 mmol) of biotin, 1.16 g (6.08 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.03 (7.6 mmol) of 1-hydroxy-1H-benzotriazole Hydrate was combined in 80 ml of dimethylformamide and stirred at room temperature for 45 min. Then 5.57 mmol of the corresponding intermediate and 2.65 ml (15.2 mmol) of Hunig base were added. After 3 h, the mixture was concentrated. The residue was purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 10/1). The appropriate fractions were combined and the solvent was evaporated in vacuo. E.1.1

[ESI-MS: m / e = 390 (M-OH ^- ) ⁺ ]. E.1.2

[ESI-MS: m / e = 420 (M-OH ^- ) ⁺ ]. E.1.3

[ESI-MS: m / e = 420 (M-OH ^- ) ⁺ ].

Educt series 2: piperazine derivatives

E.2.1

3.85 g (13 mmol) of Fmoc-glycine and 2.19 g (16.2 mmol) of 1-hydroxy-1H-benzotriazole and 2.48 g (13 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride combined in 80 ml DMF and stirred at RT for 30 min.

Then 2.01 g (10.8 mmol) of Boc-piperazine dissolved in 40 ml of DMF were added dropwise, and 2.8 g of ethyl-diisopropylamine were further added. The mixture was stirred at RT for 2 h and concentrated. The residue is taken up in dichloromethane and shaken twice with water. The organic phase was separated off, concentrated and the residue was purified by flash chromatography on silica gel (eluent: acetonitrile). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 3.91 g (78%) of the intermediate [TLC: R _f = 0.58 ²⁾ ] were obtained.

3.9 g (8.4 mmol) of this intermediate were taken up in 50 ml of dichloromethane and 25 ml of TFA were added. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether, filtered off and dried. 4.8 g (93%) of the target product E.2.1 [TLC: R _f = 0.31 ⁸⁾ ] were obtained.

E.2.2

0.53 g (3 mmol) of Boc-Glycine as well as 0.51 g (3.75 mmol) of 1-hydroxy-1H-benzotriazole and 0.58 g (3 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 40 ml of DMF and stirred at RT for 30 min. Then 1 g of ethyl diisopropylamine and then slowly 1.2 g (2.5 mmol) of product E.2.1 were added. The mixture was stirred at RT overnight and concentrated. The residue was taken up in dichloromethane and extracted twice with sodium bicarbonate solution. The organic phase was separated off, concentrated and the residue was purified by flash chromatography on silica gel (eluent: acetonitrile / water 30/1). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 760 mg (58%) of the intermediate [TLC: R _f = 0.6 ²⁾ ] were obtained.

760 mg (1.45 mmol) of this intermediate were taken up in 10 ml of dichloromethane and 5 ml of TFA were added. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether, filtered off and dried. 780 mg of the target product E.2.2 were obtained (quantitative conversion) [TLC: R _f = 0.4 ⁸⁾ ].

E.2.3

3.85 g (13 mmol) of Fmoc-Glycine as well as 2.19 g (16.2 mmol) of 1-hydroxy-1H-benzotriazole and 2.48 g (13 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 80 ml DMF and stirred at RT for 30 min. Then 2.01 g (10.8 mmol) of Boc-piperazine dissolved in 40 ml of DMF were added dropwise, and 2.8 g of ethyl-diisopropylamine were further added. The mixture was stirred at RT for 2 h and concentrated. The residue was taken up in dichloromethane and extracted twice with water. The organic phase was separated off, concentrated and the residue was purified by flash chromatography on silica gel (eluent: acetonitrile). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 3.91 g (78%) of the intermediate [TLC: R _f = 0.58 ²⁾ ] were obtained.

730 mg (1.57 mmol) of this intermediate were dissolved in 5 ml of DMF and 500 μl of piperidine were added. After stirring at RT for 15 min, the mixture was concentrated and purified by flash chromatography on silica gel (eluent ⁷⁾ ). The appropriate fractions were combined, the solvent evaporated in vacuo. The residue was stirred with diethyl ether / petroleum ether 1/1, then filtered off and the residue dried. 222 mg (58%) of the target product E.2.3 [TLC: R _f = 0.18 ¹⁰⁾ ] were obtained.

E.2.4: Piperazine- ¹³ C ₄

The amino group of glycine- ¹³ C ₂ was protected under standard conditions with the t-butoxycarbonyl (Boc) protective group.

Then 1.175 g (3.83 mmol) of Boc-Glycine- ¹³ C _{2 were dissolved} in dioxane / water 1/1 and 2.5 g (7.67 mmol) of cesium carbonate were added. It was lyophilized, the product was taken up in DMF and then 2.72 g (19.17 mmol) of iodomethane were added. After stirring at RT for 2 h, the mixture was concentrated and the residue was partitioned between dichloromethane and water. The organic phase was washed again with water, then dried over sodium sulfate and concentrated. 530 mg (72%) of the methyl ester were obtained.

525 mg (2.75 mmol) of the Boc-protected Methyl esters were then deprotected on the amino group with trifluoroacetic acid (yield: 506 mg; 90%).

The product obtained (500 mg) was converted under standard conditions with Boc-Glycine- ¹³ C ₂ to give the dipeptide conjugate, which is protected on both sides and contains four ¹³ C atoms (yield: 452 mg; 74%).

Of this intermediate product was cleaved off again with TFA the Boc group (quantitative implementation).

475 mg (1.8 mmol) of this N-terminally deblocked, ¹³ C-labeled dipeptide methyl ester trifluoroacetate were dissolved in 15 ml of methanol and 697 mg (5.4 mmol) of ethyl-diisopropylamine were added. The mixture was stirred at RT for 3 days, during which the diketopiperazine formed and precipitated. It was filtered off, washed with methanol and dried under high vacuum (yield: 138 mg; 65%).

130 mg (1.1 mmol) of the ¹³ C-labeled diketopiperazine were taken up in 40 ml of THF under argon and 284 mg (3.3 mmol) of THF-borane complex were added. The mixture was stirred under reflux for 12 h and 284 mg (3.3 mmol) of THF-borane complex were added again. After a further 16 h of reflux, the mixture was allowed to cool and quenched with 6 ml of 10% hydrochloric acid. The mixture was boiled for a further 30 min, then allowed to cool, concentrated and the residue was redistilled with dichloromethane / methanol. The residue was then washed with dichloromethane / methanol 4: 1 and filtered off. 145 mg (81%) of the fourfold ¹³ C-labeled piperazine E.2.4 [TLC: acetonitrile / water / glacial acetic acid 10/3 / 1.5: R _f = 0.23] [EI-MS: m / z = 90 (M) ⁺ radical ion].

E.2.5: Piperazine - ¹⁵ N ₂ - ¹³ C ₄

This building block was produced analogously to E.2.4 starting from glycine- ¹³ C ₂ - ¹⁵ N.

From these ¹³ C-labeled or the ¹³ C- and ¹⁵ N-labeled intermediates E.2.4 and E.2.5, all intermediate and end products of the following intermediate series and examples are made in the same way as for the unlabeled ¹² C- and ¹⁴ N analogs.

Intermediate series 1: Fully protected amino acid-flanked Piperazine derivatives

General formula:

General rule:

3 mmol of a Boc-protected Amino acid derivative and 0.51 g (3.75 mmol) of 1-hydroxy-1H-benzotriazole and 0.58 g (3 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 40 ml of DMF and added for 30 min RT stirred. Subsequently 1 g of ethyl diisopropylamine were added and then 2.5 mmol of the products E.2.1 or E.2.2 dissolved in 40 ml DMF, added dropwise. It became overnight stirred at RT and constricted. You take the backlog in dichloromethane and shake twice with sodium bicarbonate solution. The organic phase was separated, concentrated and the residue either by precipitation Dichloromethane with diethyl ether or by flash chromatography purified on silica gel (eluent: acetonitrile / water 30/1). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. The fully protected intermediates were preserved.

I.1.1 (R = H (rest of glycine))

I.1.2 (R = residue of D-valine)

Intermediate series 2: fully protected peptide-flanked Piperazine derivatives

General formula:

The Intermediate Series 2 products were manufactured according to the same general Regulation as before for the Intermediate series 1 described.

I.2.1 R = H (rest of glycine gly)))

I.2.2 (R = residue of side-chain Boc-protected histidine (His))

I.2.3 (R = residue of side chain tert-butyl ester-protected aspartic acid (Asp))

I.2.4 (R = remainder of valine (Val))

I.2.5 R = rest of asparagine (Asn))

I.2.6 (R = rest of proline (Pro))

I.2.7 (R = remainder of D-valine (D-Val))

I.2.8 (R = rest of β-alanine)

I.2.9 (R = residue of tert-butyl ester-protected glutamic acid)

Intermediate series 3: Fully protected oligo-piperazine derivatives

I.3.1.

1455 mg (11.46 mmol) oxalyl chloride were dissolved in 2 ml dichloromethane and then mixed with 100 mg (0.24 mmol) of Fmoc-piperidine in 10 ml of dichloromethane. After 1 h the solvent distilled off in vacuo and the residue redistilled with dichloromethane.

The Residue was then taken up again in 10 ml dichloromethane and made into a solution from 44 mg (0.24 mmol) Boc-piperidine and 187 mg pyridine in 10 ml Given dichloromethane. After stirring at RT for 1 h, the solvent became separated in vacuo and the residue by flash chromatography on silica gel (eluent: dichloromethane / methanol 97.5 / 2.5). The appropriate fractions were pooled and the solvent distilled off in vacuo. 74 mg (57%) of the fully protected intermediate were obtained [TLC: acetonitrile / water 20/1: Rf = 0.6].

I.3.2

1438 mg (7.72 mmol) of Boc-piperazine were dissolved in 100 ml of dichloromethane and then with 500 mg (3.86 mmol) oxalyl chloride and with 625 μl pyridine added. After stirring for 1 h was concentrated at RT and the residue stirred twice with water. The remaining solid was sufficiently pure and was in a high vacuum dried. 1230 mg (75%) were obtained.

To Standard conditions were both derived from 1230 mg of this intermediate Boc protecting groups split off (yield: 1330 mg; quantitative conversion).

305 mg (1.31 mmol) of Boc-Glycyc-glycine as well as 242 mg (1.79 mmol) of 1-hydroxy-1H-benzotriazole and 275 mg (1.43 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 25 ml of DMF and stirred at RT for 30 min. Subsequently, 543 mg (1.2 mmol) of the intermediate which had been deprotected on both sides was added dropwise dissolved in 5 ml of DMF and 625 μl of ethyl-diisopropylamine were further added. The mixture was stirred at RT for 1 h and then concentrated. The residue was purified by flash chromatography on silica gel (eluent ⁵⁾ ). The appropriate fractions were combined, the solvent was evaporated in vacuo and the residue was precipitated from dichloromethane with diethyl ether. The precipitate was filtered off and dried in a high vacuum. 276 mg (53%) of the intermediate [TLC: R _f = 0.32 ⁸⁾ ] were obtained.

182 mg (0.61 mmol) of Fmoc-Glycine as well as 124 mg (0.92 mmol) of 1-hydroxy-1H-benzotriazole and 141 mg (0.74 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride combined in 20 ml DMF and stirred at RT for 30 min. Then 2270 mg (0.61 mmol) of the intermediate described above and further 320 μl of ethyl-diisopropylamine were added. The mixture was stirred at RT for 2 h and concentrated. The residue was taken up in dichloromethane and shaken twice with water. The organic phase was separated, dried over sodium sulfate and concentrated. The residue was then precipitated from dichloromethane / methanol 1/1 with diethyl ether. The precipitate was filtered off and dried in a high vacuum. 195 mg (44%) of the intermediate I.3.1 were obtained [TLC: R _f = 0.52 ⁷⁾ ].

I.3.3

30 mg (0.123 mmol) of the educt E.2.3 were dissolved in dichloromethane, and 25 mg (0.123 mmol) of 4-nitrophenyl chloroformate were then added. After stirring at RT for 30 min, 172 μl of diisopropylethylamine are added, and after a further 30 min, 59 mg (0.123 mmol) of the educt E.2.1. Allow to stand at RT overnight and then concentrate. The residue was taken up in 20 ml of dichloromethane and shaken out with water. The organic phase was concentrated and the residue was purified by flash chromatography on silica gel (eluent ²⁾ ). The corresponding fractions were combined, the solvent was evaporated in vacuo and the residue was taken up in dioxane / water 1/1 and lyophilized. 55 mg (70%) of the intermediate I.3.2 were obtained [TLC: R _f = 0.52 ¹⁾ ] [ESI-MS: m / e = 635 (M + H) ⁺ ].

I.3.4

The Boc protective group was first split off from the compound I.3.2 in a known manner. Boc-Gly-Gly-OH was then attached in the presence of EDCI / HOBT. The target product I.3.3 was obtained in a yield of 46% over two stages. [TLC: R _f = 0.62 ⁸⁾ ]

I.3.5

47 mg (0.193 mmol) of the educt E.2.3 were dissolved in dichloromethane and then 39 mg (0.193 mmol) of 4-nitrophenyl chloroformate were added. After stirring for 10 min at RT were 269 ul Diisopropylethylamine was added and the mixture was stirred at RT for a further 20 min.

In parallel, the Boc protective group was cleaved from compound I.3.2 in a known manner. 125 mg (0.193 mmol) of the deprotected product was then added to the above approach. The mixture was stirred at RT for 4 h and then extracted with 10 ml of water. The organic phase was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The precipitate was filtered off and dried. 124 mg (80%) of the intermediate [TLC: R _f = 0.2 ⁷⁾ ] were obtained.

The Boc protective group was cleaved off from this intermediate again in a known manner. Boc-Gly-Gly-OH was then attached in the presence of EDCI / HOBT. The target product was obtained in a yield of 35% over 2 steps [TLC: acetonitrile / water / glacial acetic acid 5/1 / 0.2: R _f = 0.42] [ESI-MS: m / e = 918 (M + H ) ⁺ ].

Intermediate series 4: acid-derivatized Amino acid flanking Piperazine derivatives

General formula:

General rule:

first Step: 4.4 mmol of a series 1 intermediate were placed in 250 ml Dichloromethane was added and 50 ml of TFA were added. After 30 min stir was concentrated at room temperature and the residue from dichloromethane with Like diethyl ether, filtered off and dried.

Second step: Below argon, 0.71 mmol of this intermediate in 10 ml THF were mixed with 246.75 μl (1.42 mmol) DIEA and slowly added dropwise to a solution of 900 mg (7.1 mmol) oxalic acid dichloride in 10 ml THF. After 20 minutes, 10 ml of 1M HCl was added and the solution was stirred for a further 30 minutes. The organic phase was separated and the aqueous phase was three times. washed with 10 ml AcOEt. The corresponding organic fractions were combined and dried over MgSO ₄ . The solvent was evaporated in vacuo and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether.

I.4.1 (R = H (remainder of Glycine))

First step

Second step:

Intermediate series 5: acid-derivatized Oligo-piperazine derivatives

I.5.1

0.18 mmol of intermediate I.3.1 were taken up in 10 ml dichloromethane and mixed with 2 ml TFA. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether, filtered off and dried. The intermediate was obtained in a yield of 63.4% [TLC: acetonitrile / water / glacial acetic acid 5/1 / 0.2: R _f = 0.15] [ESI-MS: m / e = 449 (M + H) ⁺ ].

0.12 mmol of this intermediate in 5 ml and slowly added dropwise to a solution of 147 mg (1.15 mmol) of oxalic acid dichloride in 5 ml of THF under argon. 100.8 μl (0.23 mmol) DIEA were added to 5 ml THF and also slowly added dropwise. After 20 minutes, 10 ml of 1M HCl was added and the solution was stirred for a further 30 minutes. The organic phase was separated and the aqueous phase was washed three times with 10 ml AcOEt. The corresponding organic fractions were combined and dried over MgSO ₄ . The solvent was evaporated in vacuo and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The target product was obtained in a yield of 69.8% [TLC: DCM / MeOH 9/1: R _f = 0.45] [ESI-MS: m / e = 521 (M + H) ⁺ ].

Examples for acid-cleavable affinity tag

Example 1:

400 mg (0.98 mmol) of the biotin intermediate E.1.1 and 381.6 mg (1.08 mmol) of Fmoc-leucine were taken up in 8 ml DMF and cooled at -20 ° C). 188.2 mg (0.98 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added and then 6 mg (0.05 mmol) of 4-dimethylaminopyridine. The solution was stirred and slowly warmed to RT. After 5 h, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The residue was then purified by flash chromatography on silica gel (eluent: dichloromethane / methanol: 10/1). The appropriate fractions were combined, the solvent was evaporated off in vacuo and the residue was dried again from dichloromethane / methanol 1/1 with diethyl ether. 512 mg (70.2%) of the desired first intermediate were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.15].

450 mg (0.61 mmol) of this intermediate were taken up in 25 ml of DMF and 2 ml of piperidine were added. After stirring for 20 min, the mixture was concentrated and the residue was precipitated twice from dichloromethane / methanol 1/1 with diethyl ether. 287 mg (91%) of the desired second intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.05] were obtained.

50 mg (0.1 mmol) of this intermediate were taken up in 2.5 ml DMF and reacted with 102 mg (0.3 mmol) iodoacetic anhydride and 83.6 μl (0.5 mmol) DIEA. After stirring for 30 min, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The substance is then purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 10/1). After concentration, 54 mg (81.7%) of the compound according to the invention were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [FD-MS: m / e = 688 (M) ^+. ].

Example 2:

This compound according to the invention was prepared according to the same general procedure as described above for Example 1. Commercially available, labeled Fmoc leucine was used. [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [FD-MS: m / e = 695 (M) ^+. ].

Example 3:

This compound according to the invention was prepared according to the same general procedure as described above for Example 1. Only the last step is different:
400 mg (0.98 mmol) of the biotin intermediate E.1.1 and 381.6 mg (1.08 mmol) of Fmoc-leucine were taken up in 8 ml DMF and cooled at -20 ° C. 188.2 mg (0.98 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added and then 6 mg (0.05 mmol) of 4-dimethylaminopyridine. The solution was stirred and slowly warmed to RT. After 5 h, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The residue was then purified by flash chromatography on silica gel (eluent: dichloromethane / methanol 10/1). The appropriate fractions were combined, the solvent was evaporated in vacuo and the residue was dried again from dichloromethane / methanol 1/1 with diethyl ether. 512 mg (70.2%) of the desired first intermediate were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.15].

450 mg (0.61 mmol) of this intermediate was taken up in 25 ml of DMF and mixed with 2 ml of piperidine. After stirring for 20 min, the mixture was concentrated and the Residue precipitated twice from dichloromethane / methanol 1/1 with diethyl ether.

287 mg (91%) of the desired second intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.05] were obtained.

5.8 mg (0.03 mmol) 4-maleimido-butyric acid, 6.6 mg (0.03 mmol) 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.8 mg (0.04 mmol) 1- Hydroxy-1H-benzotriazole hydrate was combined in 1 ml of dimethylformamide and stirred at room temperature for 45 min. Then 0.03 mmol of the previously obtained intermediate and 15.05 μl (0.09 mmol) of Hünig base were added. After 3 h, the mixture was concentrated. The residue was purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 10/1). The appropriate fractions were combined and the solvent was evaporated in vacuo. 3.5 mg (17.7%) of the compound according to the invention were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [ESI-MS: m / e = 686 (M + H) ⁺ ].

Example 4:

This compound according to the invention was prepared according to the same general procedure as described above for Example 1. [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [ESI-MS: m / e = 719 (M + H) ⁺ ].

Example 5:

This compound according to the invention was prepared according to the same general procedure as previously described for Example 3 [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [ESI-MS: m / e = 716 (M + H) ⁺ ].

Example 6:

400 mg (0.98 mmol) of the biotin intermediate E.1.1 and 381.6 mg (1.08 mmol) of Fmoc-leucine were taken up in 8 ml DMF and cooled at -20 ° C). 188.2 mg (0.98 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added and then 6 mg (0.05 mmol) of 4-dimethylaminopyridine. The solution was stirred and slowly warmed to RT. After 5 h, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The residue was then purified by flash chromatography on silica gel (eluent: dichloromethane / methanol: 10/1). The appropriate fractions were combined, the solvent was evaporated off in vacuo and the residue was dried again from dichloromethane / methanol 1/1 with diethyl ether. 512 mg (70.2%) of the desired first intermediate were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.15].

450 mg (0.61 mmol) of this intermediate were taken up in 25 ml of DMF and 2 ml of piperidine were added. After stirring for 20 min, the mixture was concentrated and the residue was precipitated twice from dichloromethane / methanol 1/1 with diethyl ether. 287 mg (91%) of the desired second intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.05] were obtained.

6.7 mg (0.014 mmol) of the intermediate I.4.1 and 2.5 mg (0.018 mmol) of 1-hydroxy-1H-benzotriazole and 2.8 mg (0.015 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 1 ml of DMF and stirred at RT for 30 min. Then 4.8 mg (0.037 mmol) DIEA was added and then 6.4 mg (0.012 mmol) of the intermediate obtained previously. The mixture was stirred for 3 h and then concentrated. The residue was purified by precipitation from dichloromethane with diethyl ether. 3.5 mg (30%) of the desired third intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.4] were obtained.

3.5 mg (0.6004 mmol) of this intermediate were taken up in 1 ml of DMF and 20 μl of piperidine were added. After stirring for 20 min, the mixture was concentrated and the residue was precipitated twice from dichloromethane / methanol 1/1 with diethyl ether. 2.5 mg (88%) of the desired fourth intermediate were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.05].

2.5 mg (0.003 mmol) of this intermediate were taken up in 1 ml DMF and reacted with 3.3 mg (0.009 mmol) iodoacetic anhydride and 2.7 μl (0.015 mmol) DIEA. After stirring for 30 min, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The substance is then purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 10/1). 2 mg (58.7%) of the compound according to the invention were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.2]. [FABD-MS: m / e = 943 (M + H) ⁺ ].

Example 7:

400 mg (0.98 mmol) of the biotin intermediate E.1.1 and 381.6 mg (1.08 mmol) of Fmoc-leucine were taken up in 8 ml DMF and cooled at -20 ° C). 188.2 mg (0.98 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added and then 6 mg (0.05 mmol) of 4-dimethylaminopyridine. The solution was stirred and slowly warmed to RT. After 5 h, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The residue was then purified by flash chromatography on silica gel (eluent: dichloromethane / methanol: 10/1). The appropriate fractions were combined, the solvent was evaporated off in vacuo and the residue was dried again from dichloromethane / methanol 1/1 with diethyl ether. 512 mg (70.2%) of the desired first were obtained. Intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.15].

450 mg (0.61 mmol) of this intermediate was taken up in 25 ml of DMF and mixed with 2 ml of piperidine. After stirring for 20 min, the mixture was concentrated and the Residue precipitated twice from dichloromethane / methanol 1/1 with diethyl ether.

287 mg (91%) of the desired second intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.05] were obtained.

42 mg (0.081 mmol) of the intermediate I.5.1 and 16.35 mg (0.12 mmol) of 1-hydroxy-1H-benzotriazole and 18.56 mg (0.097 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride were combined in 1 ml of DMF and stirred at RT for 30 min. Then 31.3 mg (0.24 mmol) of DIEA were added and then 18.2 mg (0.089 mmol) of the intermediate product obtained previously. The mixture was stirred for 3.5 h and then concentrated. The residue was purified by precipitation from dichloromethane with diethyl ether. 35 mg (42.4%) of the desired third intermediate [TLC: dichloromethane / methanol: 10/1 R _f = 0.4] were obtained.

35 mg (0.034 mmol) of this intermediate were taken up in 5 ml of DMF and 500 μl of piperidine were added. After stirring for 20 min, the mixture was concentrated and the residue was extracted twice from dichloromethane / Me ethanol 1/1 precipitated with diethyl ether. 21 mg (76.6%) of the desired fourth intermediate were obtained [TLC: dichloromethane / methanol: 10/1 R _f = 0.05].

21 mg (0.026 mmol) of this intermediate were taken up in 1 ml of DMF and reacted with 27.8 mg (0.079 mmol) of iodoacetic anhydride and 22.8 μl (0.13 mmol) of DIEA. After stirring for 30 min, the mixture was concentrated and the residue was precipitated from dichloromethane / methanol 1/1 with diethyl ether. The substance is then purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol: 10/1). 11 mg (43.3%) of the compound according to the invention [TLC: dichloromethane / methanol: 10/1 R _f = 0.2] were obtained. [ESI-MS: m / e = 969 (M + H) ^+. ].

Analytical protein investigations

Coupling the affinity markers to SDS-7 and description of the workflow for the affinity marker from example 1

As A mixture of seven proteins was used in the sample, too Use as a size standard in gel electrophoresis (SDS-7 marker, Sigma-Aldrich GmbH, Taufkirchen).

120 μg of the protein mixture were in 5 ul Buffer 1 solved and with 135 μl Buffer 3 diluted. By heating to 100 ° C for 3 min the proteins were denatured. To reduce the existing cysteines was 3 ul reduction solution added and the mixture incubated at 100 ° C for 10 min. To implement the free cysteine with the affinity marker Example 1 was then 5 ul derivatization added and for 90 min at 37 ° C incubated.

After derivatization, 3 ul trypsin solution was added. The proteins are cleaved overnight (approx. 17 h) at 37 ° C.
Buffer 1: 50 mM Tris-HCl, pH 8.3; 5mM EDTA; 0.5% (w / v) SDS
Buffer 2: 10 mM NH ₄ acetate, pH 7
Buffer 3: 50 mM Tris-HCl, pH 8.3; 5mM EDTA
Reduction solution: 50 mM TCEP in buffer 2
Derivatization solution: 30 μg / μl affinity marker (example 1) in DMSO
Trypsin solution: 1 mg / ml trypsin (Promega GmbH, Mannheim) in buffer 3

affinity purification derivatized peptides

• – Zwei Säulenvolumina 2 × PBS
• – Vier Säulenvolumina 30 % (v/v) Acetonitril/0,4 % (v/v) Trifluoressigsäure
• – Sieben Säulenvolumina 2 × PBS
• – Vier Säulenvolumina 2 mM Biotin in 2xPBS
• – Sechs Säulenvolumina 100 mM Glycin, pH 2,8
• – Sechs Säulenvolumina 2 × PBS

The affinity columns (Monomeric Avidin, Perbio Science Deutschland GmbH, Bonn) with a column volume of 200 μl were freshly prepared before cleaning and prepared by the following washing steps:

• - Two column volumes of 2 × PBS
• - Four column volumes of 30% (v / v) acetonitrile / 0.4% (v / v) trifluoroacetic acid
• - Seven column volumes of 2 × PBS
• - Four column volumes of 2 mM biotin in 2xPBS
• - Six column volumes of 100 mM glycine, pH 2.8
• - Six column volumes of 2 × PBS

• – Sechs Säulenvolumina 2 × PBS
• – Sechs Säulenvolumina PBS
• – Sechs Säulenvolumina 50 mM Ammoniumhydrogencarbonat/20 % (v/v) Methanol
• – Ein Säulenvolumen 0,3 % (v/v) Ameisensäure

30 ul sample was diluted with 30 ul 2x PBS before application and then applied to the column. The following washing steps were then carried out to remove the non-biotinylated peptides:

• - Six column volumes of 2 × PBS
• - Six column volumes of PBS
• - Six column volumes of 50 mM ammonium hydrogen carbonate / 20% (v / v) methanol
• - A column volume of 0.3% (v / v) formic acid

• – Drei Säulenvolumina 0,3 % (v/v) Ameisensäure
• – Drei Säulenvolumina 30 % (v/v) Acetonitril/0,4 % (v/v) Trifluoressigsäure

The sample was eluted with the following steps:

• - Three column volumes of 0.3% (v / v) formic acid
• - Three column volumes of 30% (v / v) acetonitrile / 0.4% (v / v) trifluoroacetic acid

The Eluate was evaporated to dryness and only shortly before Analysis with mass spectrometry solved again.

PBS: stock solution 10x, GibcoBRL, Cat. No. 14200-067

Mass spectrometry analysis

An ion trap mass spectrometer (LCQdeka, ThermoFinnigan, San Jose) was used to analyze the peptides, which is directly connected to high pressure liquid chromatography (LC-MS). A reversed-phase column (C ₁₈ phase) was used as the separation column. The peptides were dissolved in eluent A (0.025% (v / v) trifluoroacetic acid) and injected. They were eluted with a gradient of eluent B (0.025% (v / v) trifluoroacetic acid / 84% (v / v) acetonitrile). The eluting peptides were automatically recognized by the device's acquisition software and fragmented for identification. The identity of the peptides could thus be clearly determined.

1 shows an example of a fragment spectrum of a peptide from this analysis. The observed pattern clearly identifies the peptide as the bovine serum albumin SLHTLFGDELCK sequence peptide that was part of the sample. The mass of the peptide and its fragmentation confirm that the affinity marker was cleaved by acid in the expected manner.

All in all 23 different peptides from the sample were analyzed identified, all in the same way the expected mass of the affinity brand rest. It not a single cysteine-containing peptide was identified that still complete affinity tag wore.

1 : Fragment spectrum of a peptide derivatized with the compound from Example 1 after isolation via avidin, ie with an acid-cleaved affinity marker.

Claims (20)

Connection with a protein-reactive group and an affinity ligand residue or a solid phase and a covalently linking linker, characterized in that they have the formula

in which A is the affinity ligand residue or the solid phase, S is a group of the formula

in which BO, NH or OC (= O) and is bonded to A, X is a linear or branched group of the formula (CH ₂ ) _a , in which a is a number from 1 to 10, Y is an electron-donating group and b is the number 0, 1 or 2, Z is an amino acid residue CO- (CH ₂ ) _x -CHR ^x -NH with the amino acid side chain R ^x and the number x, which is selected independently of other Z of the same or a different run variable from the range of values from 0 to 5, L 'is a bridge selected independently of other L' of the same or the other of the two run variables l and m, which enables or facilitates covalent linkage of two nitrogen atoms, Z 'each independently of the other Z 'is the same or a different run variable selected amino acid residue NH-CHR ^y - (CH ₂ ) _y -CO with the amino acid side chain R ^y and the number y from the range of values from 0 to 5, which differs from Z in the different orientation with respect to the terminal CO and NH group u distinguishes, R and R 'on one piperazine ring independently of one another and independently of other R and R' on others Piperazine rings of the same or the other of the two run variables l and m are each an α-amino acid side chain, k, l, m, n, o, p, q each independently represent a number from 0 to 10, the Sum k + l + m + n + o + p + q is at least 1 and at most 40, and PRG is the protein-reactive group, or its salt. Compound according to claim 1, characterized in that A is the affinity ligand residue or the solid phase, in the formula of the group SB O, NH or OC (= O), X is a linear group of the formula [CH ₂ ] _a , wherein a is a number from 1 to 10, Y is alkoxy or dialkylamine and b is the number 0, 1 or 2, Z is in each case the amino acid residue of glycine, leucine or alanine, independently of other Z of the same or a different variable, L 'is a bridge selected independently of other L' of the same or the other of the two run variables l and m, which is composed of building blocks selected from alkylene, alkenylene, alkynylene, arylene, CO, CS and NH, the terminal building blocks from CO and CS are selected, Z 'is the amino acid residue of glycine, leucine or alanine, independently of other Z' of the same or a different run variable, R and R 'on each piperazine ring are each the same amino acid side chain, PRG the protein-reactive Group is and the sum k + l + m + n + o + p + q is at least 1 and at most 20. Compound according to any one of the preceding claims, characterized in that A is the residue of biotin or a biotin derivative, in the formula of the group SBO or NH, X is a linear group of the formula [CH ₂ ] _a , where a is the number 1, 2 or 3, Y is alkoxy or dialkylamine and b is the number 0 or 1, Z is the amino acid residue of glycine, leucine or alanine, independently of other Z of another run variable, L 'is in each case independent of other L 'is the same or the other of the two run variables l and m selected bridge, which is composed of 2 to 10 components selected from alkylene, alkenylene, alkynylene, arylene, CO, CS and NH, the terminal components being made of CO, Z' independently of other Z 'of another run variable, the amino acid residue of glycine, leucine or alanine, R and R' are each hydrogen, -PRG is selected from

-CO- [CH ₂ ] _r -I with r = 1, 2, 3, ..., 10, -CO- [CH ₂ ] _r -Br with r = 1, 2, 3, ..., 10, -CO- [CH ₂ ] _r -Cl with r = 1, 2, 3, ..., 10 and -CO-CH = CH ₂ , and the sum k + l + m + n + o + p + q is at least 1 and at most 10. Connection according to claim 1, characterized in that Z or Z 'or Z and Z' are selected from the radicals of amino acids Glycine, leucine and alanine. Connection according to a of claims 1, 2 or 4, characterized in that A is the rest of biotin or a biotin derivative. Compound according to one of claims 1, 2, 4 or 5, characterized in that -PRG is selected from

-CO- [CH ₂ ] _r -I with r = 1, 2, 3, ..., 10, -CO- [CH ₂ ] _r -Br with r = 1, 2, 3, ..., 10, -CO- [CH ₂ ] _r -Cl with r = 1, 2, 3, ..., 10 and -CO-CH = CH ₂ . Connection according to a of claims 1, 2 or 4 to 6, characterized in that all R and R 'are hydrogen. Connection according to one of claims 1, 2 or 4 to 7, characterized in that the sum k + l + m + n + o + p + q is at most 20, in particular at most 10. Compound according to one of Claims 1, 2 or 4 to 8, characterized in that X in the formula of the group S is a linear group of the formula [CH ₂ ] _a , where a is the number 1, 2 or 3. Connection according to a the preceding claims, characterized in that B in the formula of group S is NH. Compound according to the preceding claim, characterized in that Z is the glycine residue CO-CH ₂ -NH or Z 'is the glycine residue NH-CH ₂ -CO or Z is the glycine residue CO-CH ₂ -NH and Z' is Glycine residue is NH-CH ₂ -CO. Compound according to one of the preceding claims, characterized in that L 'is selected from CO-CO, CO- (CH ₂ ) _s -CO, CO-arylene-CO, CO-CH ₂ -NH-CO-NH-CH ₂ - CO, CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO, CO-CH ₂ -NH-CO-CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO- (CH ₂ ) _s -CO-NH-CH ₂ -CO, CO-CH ₂ -NH-CO-arylene-CO-NH-CH ₂ -CO, CO and CS, where s is the number 1, 2, 3, 4, 5 or 6 is. Connection according to a the preceding claims, characterized in that they are identical with one or more or different isotopes is marked. Compound according to the preceding claim, characterized in that it is labeled with at least one carbon atom of the ¹³ C isotope. Compound according to one of the two preceding claims, characterized in that it is labeled with at least one nitrogen atom of the ¹⁵ N isotope. A process for the preparation of a compound of formula (I) according to claim 1, in which i) an affinity ligand A-OH or A-NH ₂ or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH, A-COOH or A -NH ₂ or an activated form thereof with a compound of the formula

in which B, X, Y and b have the same meaning as in formula (I), or their derivative in which the benzylic hydroxyl group bears a protective group, to give a compound of the formula

in which A, B, X, Y and b have the same meaning as in formula (II), or their derivative is reacted with a protected hydroxyl group, ii) a) a protected intermediate of the formula

in which SG and SG 'stand for two orthogonal protective groups and Z, L', Z ', R, R', k, l, m, n, o, p and q are the have the same meaning as in formula (I), the protective group SG is split off from the intermediate of the formula (IV) and the compound of the formula (III) or its protected derivative, the protective group being split off in the case of the derivative , with the deprotected intermediate of formula (IV) to a compound of formula

in A, S, Z, L ', Z', R, R ', k, l, m, n, o, p, q and SG' have the same meaning as in formulas (I), (III) and (IV), is coupled, or b) the compound of the formula (III) or its protected derivative, in the case of which derivative the protective group is first split off with a compound of the formula HO- (Z) _k - (Z) _q -SG '(VI), in which Z has the same meaning as in formula (I), k and q each independently represent a number from 0 to 10, the sum k + q being at least 1, and SG 'representing a protective group, to form a compound of the formula AS- (Z) _k - (Z) _q -SG '(VII), in which A and S have the same meaning as in formula (III) and Z, k, q and SG 'have the same meaning as in formula (VI) is coupled, or c) the compound of formula (III) or its protected derivative, in which case the protective group is first split off with a compound of the formula HO- (Z) _k -SG '(VI'), in which Z has the same meaning as in formula (I), k stands for a number from 1 to 10 and SG 'stands for a protective group, to a compound of the formula AS- (Z) _k -SG '(VII'), in which A, S and Z have the same meaning as in formula (I), k stands for a number from 1 to 10 and SG 'stands for a protective group, is coupled, an unilaterally protected intermediate of the formula

in which V represents HO or H ₂ N, Z, L ', Z', R, R 'have the same meaning as in formula (I), l, m, n, o, p, q each independently of one another Number from 0 to 10, the sum l + m + n + o + p + q is at least 1 and has at most the value of the difference 40 - k, where k is the number from 1 to 10 according to formula (VII ') and SG' stands for a protective group, is prepared from the compound of formula (VII ') the protective group SG' is split off and the compound of formula (VIII) is coupled with the deprotected compound of formula (VII ') to a compound of formula (V), where k is equal to or greater than l , and iii) from the compound of formula (V) or b) obtained in step ii) according to variant a) compound of formula (VII) or c) obtained compound of formula (V), wherein k is equal to or greater than 1 , the protective group SG 'is split off and iv) the deprotected compound obtained in step iii) finally with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula U-PRG (IX), in which U represents a group which enables the linking of PRG to the corresponding nitrogen atom of Z or a piperazine ring, and PRG has the same meaning as in formula (I). Use one or more differently labeled isotopes Connections according to one of claims 1 to 15 as a reagent for mass spectrometric analysis of proteins. Use according to the above Claim to identify one or more proteins or Protein functions in one or more protein-containing samples. Use according to a of the two preceding claims to determine the relative expression levels of one or multiple proteins in one or more protein-containing samples. Kit for mass spectrometric analysis of proteins, containing one or more differently isotope-labeled reagents Connections according to one of claims 1 to 15.