EP1446665A2 - Isotopically coded affinity markers 3 - Google Patents

Abstract

The invention concerns isotopically coded affinity markers (ICAT) for mass spectrometric analysis of proteins, and the preparation and use of said markers.

Description

Isotope-coded affinity markers 3

The invention relates to new, isotope-coded affinity markers for the mass spectrometric analysis of proteins as well as their production and their use.

Proteomics technology opens up the possibility of identifying new biological targets and markers by analyzing biological systems at the protein level. It is known that only a certain part of all possible proteins of the proteins encoded in the genome is expressed, whereby, for example, tissue type, development status, activation of receptors or cellular interactions influence the expression patterns and rates. In order to determine differences in the expression of proteins in healthy or diseased tissue, various comparative methods for analyzing protein expression patterns can be used ((a) S.P. Gygi et al., Proc. Natl.

Acad. Be. 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 differently 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)

R.H. 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 are in different

Line states have been obtained with an affinity marker of another

Isotope coding is implemented. The protein mixtures are then combined, if necessary fractionated or proteolytically treated and purified by affinity chromatography. After the bound fragments have been eluted, the eluates are analyzed by combining liquid chromatography and mass spectrometry (LC-MS). Couples or groups of with themselves only in the Isotope coding distinguishing affinity markers labeled peptides 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 (H / 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 (n) isotope effect of the differently labeled but otherwise identical ones

Peptide fragments in the LC, insufficient stability of the affinity markers in general and especially in LC-MS / MS, inefficiency of avidin monomer-based affinity chromatography, lack of flexibility in the incorporation of the isotope labels.

The object of the present invention was to provide improved isotope-coded affinity markers.

The invention relates to organic compounds suitable as affinity markers for the mass spectrometric analysis of proteins of the formula (I),

A-L-PRG (I)

in the

A for an affinity ligand residue or for a solid phase, PRG for a protein reactive group and

L stands for an A and PRG covalently linking linker,

where the linker L contains a group of the formula (! '),

in the

the amino acid residue NH-CHR ^x - (CH ₂ ) _x -CO with the amino acid side chain R ^x and the number x is in the range from 0 to 5,

is a bridge which enables or facilitates covalent linking of two piperazine residues, or in the case of 1> 2 are the same or different such bridges,

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,

Z 'is the amino acid residue CO- (CH ₂ ) _y -CHR ^y -NH with the amino acid side chain R and the number y from the value range from 0 to 5, which differs from Z in the different orientation with respect to the terminal CO- and NH group, and k, 1, m, n each independently represent a number from 0 to 10, the sum k + 1 + m + n being at least 1 and at most 40,

or their salts.

The affinity markers according to the invention have the following advantages in particular over the prior art:

Deuterium, ¹³ C- or ¹³ C- and ¹⁵ N-labeled glycine building blocks or other correspondingly labeled amino acid building blocks are inexpensive starting materials which allow the isotope-labeled affinity markers to be constructed flexibly. In the in the

Affinity markers described in formula (I) can be introduced in a simple manner more than twenty ¹³ C labels and in addition up to ten ¹⁵ N isotopes. 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 is preferred

4 differently labeled affinity markers, which enables the simultaneous analysis and relative quantification of up to 4 complex proteome samples.

The modular structure of the affinity markers enables a flexible combination of the individual components, which is adapted to the requirements of the workflow.

The affinity markers described can be equipped with an acid-labile predetermined breaking point, which enables the peptide fragments to be decomplexed by a much more efficient affinity chromatography, for example based on streptavidin or oligomeric avidin, for the biotin-modified peptide fragments or by a reversible connection to a solid phase. Furthermore, the tags remaining on the peptide fragments after acid cleavage have a low molecular weight and a high isotope density. The handling of the affinity markers is improved by improved solubility, by a crystalline or amorphous appearance and by increased stability.

The invention furthermore relates to the use of one or more differently 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 determining the relative expression levels of one or more proteins in one or more protein-containing samples.

The invention further relates to a kit for the mass spectrometric analysis of proteins, containing as reagents one or more differently isotope-labeled compounds according to the invention.

An affinity ligand A is used for the selective enrichment of samples using the

Affinity chromatography. The affinity columns are provided with the corresponding complementary partners to the affinity ligands, which enter into covalent or non-covalent bonds with the affinity ligands. An example of a suitable affinity ligand is biotin or a biotin derivative which is strong, non-covalent with the complementary peptides avidin or streptavidin

Binds. In this way, samples to be examined can be selectively isolated from sample mixtures with the aid of affinity chromatography. In the same sense, carbohydrate residues, for example, which can enter into non-covalent interactions with, for example, fixed lectins, can also be used as an affinity ligand. Furthermore, in the same sense

Interaction of haptens with antibodies or the interaction of transition metals with corresponding ligands can be used as complexing agents or other systems that interact with each other.

Alternatively, the targeted depletion can also be carried out by selective, reversible

Connection to an appropriately functionalized fixed phase A can be achieved. Suitable solid phases are, for example, Ainino-functionalized resins based on silica gel and are furthermore 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):

Preferred as solid phase A is a polymeric carrier, in particular a modified natural or synthetic resin, for example a resin based on silica gel or polyethylene glycol, with functional groups suitable for linking the linker L, for example hydroxyl, carboxy and amino groups, in particular amino groups , Particularly preferred as solid phase A is an amino-functionalized resin based on silica gel, for example an aminopropyl silica gel, as described, for. B. is sold by Aldrich under number 36425-8.

Protein-reactive groups PRG are used for the targeted labeling of proteins on selected functional groups. PRGs have a specific reactivity for terminal functional groups of the proteins. Amino acids as elements of proteins that are often used for targeted labeling are, for example, mercaptoaminomonocarboxylic acids such as cysteine, diaminomono- carboxylic acids such as lysine or arginine or monoaminodicarboxylic acids such as aspartic acid or glutamic acid. Protein-reactive groups can also be phosphate-reactive groups such as metal chelates and aldehyde-reactive and ketone-reactive groups such as semicarbazones or amines with subsequent treatment with sodium borohydride or sodium cyanoborohydride. It can also be groups which react with the reaction products after targeted protein derivatization, such as, for example, a bromine cyanide cleavage or an elimination of phosphate groups etc.

Z and Z 'are residues of the same or different amino acids. Residues of L-α-amino acids, in particular the 20 natural proteinogenic amino acids (x = 0 or y = 0), for example glycine residues, and residues of ω-amino acids, such as NH- (CH ₂ ) ₂ -CO, are preferred and CO- (CH ₂ ) ₂ -NH. The amino acids can optionally be in the D, L or racemic form.

Preferred compounds of the formula (I) according to the invention have a group of the formula (I ") in the linker L as a group of the formula (T) (all R and R 'in formula (V) are hydrogen):

Likewise preferred compounds of the formula (I) according to the invention have in

Linker L is a group of formula (T), in particular a group of formula (I "), in which Z is the glycine residue NH-CH ₂ -CO or Z 'is the glycine residue CO-CH ₂ -NH, in particular in the Z the glycine residue NH-CH ₂ -CO and Z 'the glycine residue CO-CH ₂ -

NH is. In a further embodiment, linkers L are preferred which have a predetermined breaking point S such as, for. B. have an acid-labile functionality, which ensure cleavage of the affinity marker under certain conditions, for example under the action of acid, so as to. B. to facilitate the release of the affinity column, or to reduce the size of the residue remaining on the peptide, or to make the work processes more efficient overall. Instead of an acid-labile predetermined breaking point, the linker L can also be cleaved in another way, for example by chemical cleavage of, inter alia, silyl ethers, esters, carbamates, thioesters, acetals, disulfides or Schiff bases, furthermore by photochemical cleavage, or by enzymatic cleavage of Esters, amides, nucleotides or glycosides or by thermal cleavage of, for example, interacting nucleic acid strands.

To improve the solubility of the affinity markers according to the invention, existing acidic and / or basic functional groups in the form of their salts, preferably their hydrochlorides, acetates, trifluoroacetates, alkali metal salts or ammonium salts, can be prepared and used.

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

Preferred compounds according to the invention are those of the formula (I), in particular of the formula (II),

in which one or more of the groups A, PRG, S, Z, L ', Z' and k, 1, m, n, in particular all of these groups, are selected in accordance with the following definitions:

A stands for the acyl radical of an affinity ligand, for example biotinyl or a biotin derivative, or for a functional group connected to a polymeric carrier, for example a carrier-bound hydroxyl, carboxy or amino group, in particular a carrier-bound amino group.

PRG stands for the rest of a protein-reactive group, which is characterized by an electrophilic group and a suitable bridge which enables or facilitates the attachment of the electrophilic group to Z '. Furthermore, such a group can also improve solubility, for example, by appropriate design. A preferred protein-reactive group is an epoxide, a maleimido group, a halogen or an acrylic radical, in particular bridged electrophiles such as

-CO- [CH ₂ ] _r -Cl with r = 1-10, -CO-CH = CH ₂ .

Furthermore, PRG can be another known protein-reactive group, e.g. B. by WH Scouten in Methods in Enzymology, Volume 135, edited by Klaus Mosbach, AcademicPress Inc. 1987, p. 30ff.

S stands for an acid-labile predetermined breaking point, such as

with Y as a spacer with preferably 1-10, in particular 1-5, non-hydrogen atoms, which enables or facilitates the connection of the aryl radical to the affinity ligand or to the polymeric support, particularly preferably NH, NH-CH ₂ , NH-CH ₂ -CH ₂ -NH-CO, CH ₂ -CO, where Y can be ortho, meta and para to NH and the para position is preferred,

and with SK as the side chain residue of an α-amino acid of the formula SK-CH (NH ₂ ) -COOH, which can be present in the D, L or racemic form for other SK as an H atom, for example the side chains of the 20th natural amino acids as well as their D-forms and racemates.

Z is the residue of an amino acid, especially the residue of

Glycine, which may not be labeled or may contain ¹³ C or ¹⁵ N labels, or a combination of these labels.

L 'is a bridge that is a covalent link between two

Piperazine residues enabled or relieved. L 'is preferably built up from the building blocks alkylene, in particular (CH ₂ ) _S , alkenylene, alkynylene, axylene, CO, CS and NH, in particular from a number of 2 to 10 such blocks. V may contain further amino acid residues such as Z and Z ', in particular glycine residues, which may optionally contain ¹³ C or ¹⁵ N isotope labels or a combination of these labels, and is preferably a bridge selected from

CO-CO, CO- (CH ₂ ) _s -CO and CO-arylene-CO, CO-CH ₂ -NH- CO-NH-CH ₂ -CO, CO-NH-CH ₂ -CO, CO-CH2-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, (CH ₂ ) _s -NH-CO-CO-NH- (CH ₂ ) _s , (CH ₂ ) ₃ -NH-CO-CO-NH-

(CH ₂ ) ₃ , (CH ₂ ) _s -CO, (CH ₂ ) ₂ -CO, CO and CS, where s is preferably an integer between 1 and 6, for example 2, 3, 4 or 5.

and 'on a piperazine ring are the same amino acid

Side chains, in particular hydrogen, are preferably identical amino acid side chains, in particular hydrogen, on all piperazine rings of one of the two run variables 1 and m, and are particularly preferably identical amino acid side chains, in particular hydrogen, on all piperazine rings.

Z 'is the residue of an amino acid, especially the residue of

Glycine, which differs from Z in the different orientation with respect to the terminal CO and NH groups; it may not be labeled or contain ¹³ C, or ¹⁵ N labels, or a combination of these labels.

k, 1, m, n can each independently stand for numbers between 0 and 10, the sum of k + 1 + m + n preferably being greater than 0 and less than 20 and particularly preferably being less than 10. M is preferably the number 0 or 1. In the context of the present invention, unless otherwise specified, alkyl, alkylene, alkenylene, alkynylene, alkoxy and arylene have the following meaning:

Alkyl per se and "alk" and "alkyl" in alkylene, alkenylene, alkynylene and alkoxy stand for a linear or branched alkyl radical with generally 1 to 6, preferably 1 to 4, particularly preferably 1 to 3 carbon atoms, by way of example and preferably for Methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.

Alkylene, alkenylene and alkynylene represent divalent alkyl groups which, in the case of alkenylene or alkynylene, have one, two, three, four, five or more double or triple bonds and accordingly have at least 2 carbon atoms, for example and preferably for methylene, ethylene, ethenylene , Ethynylene, n-

Propylene, iso-propylene, n-propenylene, methylethenylene and propinylene.

Alkoxy is exemplary and preferably methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Arylene stands for a divalent mono- to tricyclic aromatic, carbocyclic radical with usually 6 to 14 carbon atoms; exemplary and preferably for phenylene, naphthylene and phenanthrenylene.

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

The non-isotope-labeled compounds already represent isotope coding. The compounds according to the invention are used for further isotope coding preferably isotopically labeled with at least one carbon atom of the ¹³ C isotope, 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. Alternatively or additionally, the isotopes D, N, O, O and / or can also be used for labeling

34 S can be used.

In a particular embodiment of the invention, 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 three ¹⁵ N atoms.

The isotope markings are usually carried out in L and / or PRG, preferably in L and there in particular in groups Z, L ', Z' and / or in the piperazine-B blocks.

The compounds according to the invention can be prepared, for example, by firstly protecting an intermediate of the formula (III) which is protected with suitable orthogonal protective groups SG and SG ',

SG- (Z) _k (ZVSG '(in)

for example with the Boc and Fmoc groups according to formula (IQ '),

(IIP) will be produced. The synthesis of these intermediates proceeds according to classic methods of peptide chemistry as are known to the person skilled in the art and as described, for example, in Houben-Weyl; Methods of organic chemistry; Fourth edition; Volume XV parts 1 and 2; Georg Thieme Verlag Stuttgart 1974, or in Hans-Dieter Jakubke and Hans Jeschkeit: amino acids, peptides, proteins; Verlag Chemie,

Weinheim in 1982.

From these intermediates, the protective group SG is first detached again after standard reactions and then, if appropriate, a further amino acid derivative which carries an identical or different protective group SG, in particular a Boc group, to the amino function, is attached, derivatives of the formula (IV ) can be obtained. Third party functions available in SK may optionally be protected or freely available. Protective groups that are optionally present can be retained permanently or in separate deblocking steps or simultaneously with the splitting off of one of the terminal groups

Protection groups are replaced.

(IV)

SG-NH-CH (SK) -CO- (Z) _k

The protective group SG ', for example an Fmoc group with piperidine in DMF, can then be removed from (IN), so that in the next step the coupling 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 (V)

with U a group which enables the linkage of PRG with Z 'or possibly with another end group of L, for example by becoming a leaving group. Examples of such groups are activated Esters such as B. N-hydroxysuccinimide ester or chlorides or groups from which a leaving group can be generated during the coupling.

In a further step, the terminal protective group SG is then removed, a conjugate of the formula (VI) being obtained.

H ₂ N-CH (SK) -CO- (Z) _k (Z ') _n -PRG (VI)

In parallel, an affinity ligand A-OH or A-NH ₂ or an activated form of the same such as, for example, an activated ester, an acid chloride or the like, or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH or A-NH ₂ or an activated form thereof under suitable coupling conditions with a compound

which can optionally also carry a protective group, for the derivative

implemented. The derivative (VIII) is then, if appropriate after prior splitting off of an optionally introduced protective group with activated carbonic acid derivatives such as thiophosgene or thiocarbonylbisimidazole, converted into a corresponding isothiocyanate and then coupled with (VI) to the thiourea (IX). NH-CH (SK) -CO- (Z) _k (Z ') π-PRG (IX)

For example, the amino function on resins can first be reacted with Fmoc-protected p-aminobenzoic acid or with Fmoc-protected p-aminophenylacetic acid, then the Fmoc group can be removed and converted into the isothiocyanate with an activated carbonic acid derivative and converted to the thiourea.

Another object of the invention is accordingly a method for producing a compound according to claim 1, in which

i) a protected intermediate of the formula (III),

where SG and SG 'represent two orthogonal protecting groups,

will be produced,

ii) the protective group SG is first detached from the intermediate of the formula (in) and then a further amino acid derivative which carries an identical or different protective group SG to the α-amino function which is detached is attached, with a derivative of the formula (IV) . SG-NH-CH (SK) -CO- (Z) _k (Z SG '(IV)

in which SK stands for the side chain of an amino acid,

be obtained

iii) after detachment of the protective group SG 'from the derivative of the formula (IV) with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula (V),

U-PRG (V)

in which U stands for a group which enables PRG to be linked to Z 'or, if appropriate, to another end group of L,

is implemented

iv) the terminal protective group SG is removed, a conjugate of the formula (VI)

H ₂ N-CH (SK) -CO- (Z) _k (Z ') _n -PRG (VI)

will get v) an affinity ligand A-OH or A-NH or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH or A-NH ₂ or an activated form thereof with a compound of formula (VII),

in which Y stands for the optionally branched spacer group,

which can optionally carry a protective group, to the derivative of the formula (VIII)

is implemented

vi) the derivative of the formula (VIII) is then converted into a corresponding isothiocyanate after prior splitting off of an optionally introduced protective group,

vii) the isothiocyanate is then coupled with the conjugate of the formula (VI) to the thiourea of the formula (IX) and

vii) any protective groups still present are split off in an optional last step, the successive steps v) and vi) can be carried out at any time before step vii).

In all reaction steps, reversible cleavable protective groups, as is customary in peptide chemistry, can be used. A protective group SG can be retained, or can be removed simultaneously with the Boc protective group or can be removed in a separate step. Suitable protective groups are, for example, the Boc protective group which can be cleaved with trifluoroacetic acid or the Fmoc protective group which can be cleaved with piperidine or morpholine. Other suitable protecting groups and the corresponding methods for introduction and separation were z. B. described 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.

Optionally, the affinity markers can also be built up in reverse order, the Boc protective group being first detached from derivatives of the formula (IV). The deblocked compounds are then reacted with isothiocyanates correspondingly generated from (Vffl) to give compounds of the formula

After detaching the Fmoc protective group with piperidine, the coupling with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group is carried out in the last step

U-PRG (V)

made and thus also compounds of formula (IX) obtained. The invention accordingly furthermore relates to a method for producing a compound according to claim 1, in which

i) a protected intermediate of the formula (III),

SG- (Z) _k (Z ') _n -SG' (III)

in which SG and SG 'stand for two orthogonal protective groups,

will be produced,

ii) the protective group SG is first detached from the intermediate of the formula (III) and then a further amino acid derivative which bears an identical or different protective group SG to the -amino function is attached, a derivative of the formula (IV )

SG-NH-CH (SK) -CO- (Z) _{k (Z} ') _n -SG' (IV)

in which SK stands for the side chain of an amino acid,

be obtained

iii) the terminal protective group SG is detached, a conjugate of the formula (VT) (VI ')

H ₂ N-CH (SK) -CO- (Z) _k

will get

iv) an affinity ligand A-OH or A-NH ₂ or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH or A-NH ₂ or an activated form thereof with a compound of formula (VII),

in which Y stands for the optionally branched spacer group,

which can optionally carry a protective group to the derivative of the formula (VQI)

is implemented

v) the derivative of the formula (VIII) is then converted into a corresponding isothiocyanate after prior splitting off of an optionally introduced protective group,

vi) the isothiocyanate is then coupled with the conjugate of the formula (VT) to the thiourea of the formula (X ') and

vii) after detachment of the protective group SG 'from the thiourea of the formula (X') with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula (V),

U-PRG (V)

in which U stands for a group which enables PRG to be linked to Z 'or, if appropriate, to another end group of L,

is implemented and

viii) in an optional last step, any protective stacks still present are split off,

the successive steps iv) and v) can be carried out at any time before step vi).

The reactions can be carried out at various pressure and temperature ratios, for example at 0.5 to 2 bar and preferably under normal pressure, or -30 to +100 ° C and preferably -10 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. Usually are

Reaction in DMF, dichloromethane, THF, dioxane / water or THF / dichloromethane at room temperature or under ice cooling and normal pressure is preferred. Thus, the described types of affinity markers can be produced in various ways, both linearly and by coupling blocks which have already been prepared. The modular construction principle enables a multitude of conceivable combinations and, with the appropriate selection of isotope-labeled modules, any type, number and placement of the isotope markings. In molecules of the same type, any number, preferably up to 30, of isotope marker ranges can only be realized with the inclusion of C and N isotopes. This opens the way to simultaneous analysis of several, preferably up to 4, complex protein samples.

Examples

42 affinity markers of formula (II), in which A is the acyl residue of biotin, and one affinity marker of formula (II), in which A is a polymer carrier functionalized with amino groups, (example 42) were prepared and are described in US Pat summarized below table. With the exception of Z in Example 26 and Z 'in Example 43, Z and Z' stand for the respective glycine residue, with the exception of Z, so that in each case only the values of k and n are given and for (Z in Example

1

26 the group Z and for (Z ') _n in example 43 the group Z is specified and defined.

Used abbreviations:

Y. - Yield

Boc - tert-butoxycarbonyl

DIEA - Diisopropylethylamine (Hünig's base)

DMAP - dimethylaminopyridine

DMF - dimethylformarnide DMSO - dimethyl sulfoxide

EI - electron impact ionization

ESI - electrospray ionization

Fmoc - fluorenyl-9-methoxycarbonyl

HPLC - High-performance liquid chromatography MALDI - Matrix-assisted laser desorption ionization

MS mass spectrometry

MTBE - methyl tert-butyl ether quant. - quantitative (i.e. full) implementation

RP - Reverse phase RT - room temperature

TCEP - triscarboxyethylphosphine TFA - trifluoroacetic acid

THF - tetrahydrofuran

TLC - thin layer chromatography

(v / v) - Concentration in volume per volume (w / v) - Concentration in mass per volume

Unless expressly stated otherwise, the composition of solvent and eluent mixtures is stated by specifying the components separately from "/" and reproducing the relative parts by volume. So z. B. "Acetonitrile / water 10/1" a mixture of acetonitrile and water in

Volume ratio of 10 to 1.

Preferred eluents (reference by specifying ^l 'etc.):

^1} acetonitrile / water 10/1

²⁾ Acetonitrile / water 20/1

³⁾ dichloromethane / methanol 97.5 / 2.5

⁴⁾ Acetonitrile / water / glacial acetic acid 10/1 / 0.1

⁵⁾ acetonitrile / water / glacial acetic acid 5/1 / 0.2 ⁶⁾ acetonitrile / water / glacial acetic acid 10/3 / 1.5

⁷⁾ dichloromethane / methanol / aq. Ammonia (17%) 15/2 / 0.2

⁸⁾ Acetonitrile / water / glacial acetic acid 10/5/3

⁹⁾ dichloromethane / methanol / aq. Ammonia (17%) 15/4 / 0.5

For the preparation of the exemplary affinity markers described below, the biotin derivatives of the educt series 1 and the piperazine derivatives of the educt series 2 were first prepared. The piperazine derivatives were then converted into the intermediates of the intermediate series 1 to 3. These intermediates were then implemented with the biotin derivatives to the corresponding affinity markers. Educt series 1: biotin derivatives

E.l.l

1 g (4.09 mmol) of biotin, 500 mg (4.09 mmol) of 4-aminobenzylamine and 830 mg (6.14 mmol) of 1-hydroxy-1H-benzotriazole, 942 mg (4.91 mmol) of N- (3rd -Dimethyl-aminopropyl) -N'-ethylcarbodiimide hydrochloride and 1587 mg of ethyl-diisopropyl-amine were combined in 40 ml of DMF. It got over night

Stirred at room temperature, concentrated and the residue purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol / aqueous ammonia (17%) 15/3 / 0.3). The appropriate fractions were combined and the solvent evaporated in vacuo. The residue was stirred with diethyl ether and suction filtered. 1097 mg (77%) of the intermediate were obtained

[TLC: dichloromethane / methanol / aq. Ammonia (17%) 15/4 / 0.5: R _f = 0.58] [ESI-MS: m / e = 349 (M + H) ⁺ ].

600 mg (1.72 mmol) of this intermediate were dissolved in 40 ml of dioxane / water 1/1 and 298 mg (2.58 mmol) of thiophosgene and 890 mg of ethyl-diisopropylamine were added. The mixture was stirred at room temperature for 10 min and concentrated. The target product E.l.l was precipitated from dichloromethane / methanol with diethyl ether.

Yield: 616 mg (92%) [TLC: R _f = 0.56 ⁵⁾ ] [ESI-MS: m / e = 391 (M + H) ⁺ ]. E.1.2

Mono-Fmoc-protected p-phenylene diamine was prepared using standard methods such as those described in e.g. B. in Houben-Weyl; Methods of organic chemistry; Fourth edition; Volume XV parts 1 and 2; Georg Thieme Verlag Stuttgart 1974, or in Hans-Dieter Jakubke and Hans Jeschkeit: amino acids, peptides, proteins; Verlag Chemie, Weinheim 1982 are produced.

200 mg (0.82 mmol) of biotin were taken up in 10 ml of dichloromethane and 974 mg (8.2 mmol) of thionyl chloride were added. After stirring for 1 h, the mixture was concentrated and redistilled twice with dichloromethane.

The resulting acid chloride (0.81 mmol) was taken up in 30 ml of dichloromethane and 387 mg (4.9 mmol) of pyridine and 242 mg (0.55 mmol) of mono-Fmoc-protected p-phenylene diamine were added. The mixture was stirred at RT for 2 days and the precipitated product was filtered off. 300 mg (99%) of the intermediate were obtained, which was used in the next reaction step without further purification [TLC: R _f = 0.5 ¹⁾ ].

The crude product was taken up in 5 ml of DMF and 500 μl of piperidine were added. After stirring for 15 min at room temperature, the mixture was concentrated and the residue was purified by flash chromatography on silica gel (elution mixture ⁷⁾ ). The appropriate fractions were combined, the solvent removed and the

Residue dried in vacuo. 69 mg (39%) of the deprotected intermediate were obtained. 65 mg (0.19 mmol) of this intermediate were dissolved in 10 ml of dioxane / water 1/1 and 33 mg (0.29 mmol) of thiophosgene and 100 mg of ethyl-diisopropylamine were added. The mixture was stirred at room temperature for 10 min and concentrated. The target product E.1.2 was precipitated from dichloromethane / methanol with diethyl ether.

Yield: 65 mg (89%) [TLC: R _f = 0.43 ^1} ] [ESI-MS: m / e = 377 (M + H) ⁺ ].

E.1.3

500 mg (3.65 mmol) of 4-amino-benzoic acid were taken up in 20 ml of DMF and with 872 mg (2.73 mmol) of the hydrochloride of the mono-Fmoc-protected ethylenediamine and with 739 mg (5.47 mmol) 1- Hydroxy-1H-benzotriazole and treated with 839 mg (4.38 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride. The mixture was stirred at room temperature for 2 h, concentrated and the residue was taken up in 200 ml of dichloromethane. It was shaken out three times with 200 ml of sodium hydrogen carbonate solution. The organic phase was concentrated and the residue was purified by flash chromatography on silica gel (eluent: acetonitrile). The appropriate fractions were combined and the solvent evaporated in vacuo and the residue dried. 639 mg (59%) of the intermediate product were obtained [TLC: R _f = 0.68 ²⁾ ]

400 mg (1 mmol) of the intermediate were taken up in 10 ml of DMF and 500 μl of piperidine were added. After stirring for 15 min at room temperature, the mixture was 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, the solvent was removed and the residue was dried in vacuo. 147 mg (82%) of the deprotected intermediate were obtained [TLC: R _f = 0.18 ⁵⁾ ].

191 mg (0.78 mmol) biotin was taken up in 10 ml DMF and with 158 mg

(1.17 mmol) 1-hydroxy-1H-benzotriazole and 180 mg (0.94 mmol) N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride. The mixture was stirred at RT for 10 min and then 303 mg of ethyldiisopropylamine and 140 mg (0.78 mmol) of the deprotected intermediate were added. The mixture was then stirred at RT for a further 6 h and concentrated, and the crude product was precipitated from dichloromethane with diethyl ether. The

The residue was separated off and purified by flash chromatography on silica gel (elution mixture: dichloromethane / methanol / aqueous ammonia (17%) 15/3 / 0.3). The appropriate fractions were combined and the solvent evaporated in vacuo and the residue dried. 222 mg (70%) of the intermediate were obtained [TLC: dichloromethane / methanol / aq. ammonia

(17%) 15/4 / 0.5: R _f = 0.47].

200 mg (0.54 mmol) of this intermediate were dissolved in 15 ml of dioxane / water 1/1 and 94 mg (0.81 mmol) of thiophosgene and 210 mg of ethyl-diisopropylamine were added. The mixture was stirred at room temperature for 15 min and concentrated. The

The target product was precipitated from dichloromethane / methanol with diethyl ether.

Yield: 230 mg (95%) [TLC: R _f = 0.44 ⁵⁾ ] [ESI-MS: m / e = 448 (M + H) ⁺ ].

Educt series 2: piperazine derivatives

E.2.1

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'- ethyl carbodiimide hydrochloride were combined in 80 ml of 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. It was rubbed at RT for 2 h and concentrated. The residue was 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 pooled

Evaporated solvent in vacuo and the residue dried. 3.91 g (78%) of the intermediate product were obtained [TLC: R _f = 0.58 ²⁾ ].

3.9 g (8.4 mmol) of this intermediate were taken up in 50 ml dichloromethane and treated with 25 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. 4.8 g (93%) of the target product E.2.1 [TLC: R _f = 0.31 ⁵⁾ ] 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 hydrogen carbonate 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 ⁵⁾ ]. 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 of 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 product were obtained [TLC: R _f = 0.58 ²⁾ ].

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

The residue was stirred with diethyl ether / petroleum ether 1/1, then filtered off and the residue was dried. 222 mg (58%) of the target product E.2.3 [TLC: R _f = 0.18 ^{7) were obtained} . E.2.4: Piperazine- ¹³ C ₄

The amino group of glycine- ¹³ C ₂ was protected according to 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 ester 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 form the dipeptide conjugate, which is protected on both sides and contains four ¹³ C atoms (yield: 452 mg; 74%).

The Boc grappa from this intermediate was again replaced with TFA (quantitative conversion).

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, the Diketopiperazine formed and failed. It was filtered off, washed with methanol and dried in a 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 refluxed 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 four-fold 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 were obtained (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 ¹ 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 were produced in the same way as for the unlabeled ¹² C- and ¹⁴ N analogs. E.2.6

7.459 g (24.36 mmol) of benzyloxycarbonyl-glycine-N-hydroxysuccinimide ester in 70 ml of DMF were combined with 3 g (34.83 mmol) of piperazine and ethyldiisopropylamine and stirred at RT for 2 h. The mixture was then concentrated and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane / methanol / aqueous ammonia (17%) 15/2 / 0.2). The appropriate fractions were combined, the solvent evaporated in vacuo and the

Residue dried. 4.04 g (60%) of the target compound were obtained [TLC: R _f = 0.26 ⁵⁾ ].

E.2.7

The Boc protective group was introduced into the compound E.2.5 according to standard conditions with 0.5 equivalents of Boc anhydride. Yield: 65% [TLC: R _f = 0.22 ⁵⁾ ]. E.2.8

The Fmoc protective group was first introduced into the compound E.2.7 using Fmoc-Cl according to standard conditions and then the Boc protective group was removed using trifluoroacetic acid. [TLC: R _f = 0.32 ⁵⁾ ].

E.2.9

The protected dipeptide bearing two ¹⁵ N and three ¹³ C labels was produced according to standard conditions over 5 steps: First, the Z protection was made in [Bis- ¹³ C, ¹⁵ N] - glycine with benzyloxycarbonyl chloride in dioxane / IN sodium hydroxide solution. group introduced (yield: 47%). This amino acid derivative was coupled with [bis- ¹³ C] glycine methyl ester (intermediate in the preparation of E.2.4) (yield: 77%) and in the last step the methyl ester was cleaved with 2N lithium hydroxide in methanol (yield: 75%). [TLC: R _f = 0.25 ⁴⁾ ] [ESI-MS: m / e = 272 (M + H) ⁺ ]. E.2.10

Manufactured analogously to E.2.6 or from Fmoc-Pip.

[TLC: R _f = 0.35 ⁵⁾ ].

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 stirred at RT for 30 min. Then 1 g of ethyl-diisopropylamine was added and then 2.5 mmol of the products E.2.1 or E.2.2 dissolved in 40 ml of DMF were added dropwise.

The mixture was stirred at RT overnight and concentrated. The residue was taken up in dichloromethane and shaken twice with sodium bicarbonate solution. The organic phase was separated off and concentrated, and the residue was purified either by precipitation from dichloromethane with diethyl ether or 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. The fully protected were preserved

Intermediate.

1.1.1 (R = H (rest of glycine))

Educts: Boc-Glycine; E.2.1

Yield: 58% R _f = 0.45 ^1}

1.1.2 (R = remainder of D-valine)

Educts: Boc-D-valine; E.2.1

Purification: flash chromatography; Eluent: dichloromethane / methanol 98/2 yield: 76% R _f = 0.3 ³⁾

Intermediate series 2: fully protected peptide-flanked piperazine derivatives

General formula:

The intermediate series 2 products were manufactured according to the same general procedure as previously described for the intermediate series 1.

1.2.1 (R = H (rest of glycine (Gly)))

Educts: Boc-Glycine; E.2.2

Yield: 86% R _f = 0.55 ^1}

1.2.2 (R = residue of side-chain Boc-protected histidine (His))

Educts: Bis-Boc-histidine-N-hydroxysuccinimide ester; E.2.2

Special features: Instead of the EDCI / HOBT activation rank, the

Hydroxy-succinimide ester used. Purification by precipitation. Yield: 85% R _f = 0.47

1.2.3 (R = residue of side chain tert-butyl ester-protected aspartic acid (A p))

Educts: Boc-aspartic acid-γ-tert-butyl ester; E.2.2 Special features: cleaning by precipitation.

Yield: 68% R _f = 0.66 ^υ

1.2.4 (R = remainder of valine (Val))

Educts: Boc-valine; E.2.2

Special features: cleaning by precipitation. Yield: 79% R _f = 0.64 ⁴⁾

1.2.5 (R = residue of asparagine (Asn))

Educts: Boc asparagine; E.2.2 Special features: Purification by flash chromatography with eluent ²⁾ Yield: 66% R _f = 0.47 ¹⁾

1.2.6 (R = rest of proline (Pro))

Educts: Boc-Proline-N-hydroxysuccinimide ester; E.2.2

Special features: The activated amino acid derivative was used instead of EDCI / HOBT. Purification by flash chromatography with

Acetonitrile / water 15/1. Yield: 98% R _f = 0.55 ⁵⁾

1.2.7 (R = remainder of D-valine (D-Val))

Educts: Boc-D-valine; E.2.2 Special features: Purification by flash chromatography with acetonitrile / water 30/1

Yield: 80% R _f = 0.64 ⁾

1.2.8 (R = remainder of ß-alanine)

Educts: Boc-ß-alanine; E.2.1

Special features: First E.2.1 was implemented with Boc-ß-alanine, then the Boc-

Grappe replaced, and finally Boc-Glycine attached. Yield: 15% over 3 stages R _f = 0.45 ⁷⁾

1.2.9 (R = residue of tert-butyl ester-protected glutamic acid)

Educts: Boc-glutamic acid-γ-tert-butyl ester; E.2.2

Yield: 98% R _f = 0.75 Intermediate series 3: oligo-piperazine derivatives

1.3.1

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

According to standard conditions, both Boc protection groups were removed from 1230 mg of this intermediate (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'- ethyl carbodiimide 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, dissolved in 5 ml of DMF, were added dropwise, and 625 μl of ethyldiisopropylamine 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 ⁵⁾ ] 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'-ethylcarbo- diimide hydrochloride were combined in 20 ml DMF and stirred at RT for 30 min. Then 2270 mg (0.61 mmol) of that described above

Intermediate and added 320 ul ethyl-diisopropylamine. 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

The precipitate was filtered off and dried under high vacuum. 195 mg (44%) of the intermediate 1.3.1 [TLC: R _f = 0.52 ⁴⁾ ] were obtained.

1.3.2

30 mg (0.123 mmol) of the educt E.2.3 were dissolved in dichloromethane, and 25 mg (0.123 mmol) of 4-mtrophenyl chloroformate were then added. After stirring at RT for 30 min, 172 μl of diisopropylethylamine were added and, after a further 30 min, 59 mg (0.123 mmol) of the educt E.2.1. The mixture was left at RT overnight and then concentrated. 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 appropriate fractions were combined, the solvent in vacuo evaporated and the residue taken up in dioxane / water 1/1 and lyophilized. 55 mg (70%) of the intermediate 1.3.2 [TLC: Rf = 0.52 °] [ESI-MS: m / e = 635 (M + H) ⁺ ] were obtained.

1.3.3

368 mg (0.63 mmol) of the intermediate 1.2.1 were taken up in 5 ml of DMF and 500 μl of piperidine were added. After stirring for 15 min at room temperature, the mixture was concentrated and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane / methanol / aqueous ammonia (17%) 15/3 / 0.3). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 204 mg (90%) of the target product Boc-Gly-Gly-Pip-Gly [TLC: dichloromethane / methanol / aq. ammonia

(17%) 15/4 / 0.5: R _f = 0.28].

70 mg (0.196 mmol) of this intermediate were dissolved in 18 ml of dichloromethane and then treated with 59 mg (0.294 mmol) of 4-nitrophenyl chloroformate. After stirring at RT for 10 min, 273 μl of diisopropylethylamine and after a further 2 h

105 mg (0.196 mmol) of compound E.2.2 were added. The mixture was stirred at RT overnight. A solid precipitated out and was filtered off. 15 mg (10%) of the intermediate 1.3.3 [TLC: R _f = 0.18 ⁴⁾ ] [ESI-MS: m / e = 806 (M + H) ⁺ ] were obtained, 1.3.4

The Boc protection group was first replaced in a known manner from connection 1.3.2. Then Boc-Gly-Gly-OH was in the presence of

EDCI / HOBT attached. The target product 1.3.4 was obtained in a yield of 46% over two steps. [TLC: R _f = 0.62 ⁵⁾ ]

1.3.5

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

In parallel, the Boc protective group was detached from compound 1.3.2 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 The precipitate was filtered off and dried. 124 mg (80%) of the intermediate [TLC: R _f = 0.2 ⁴⁾ ] were obtained.

The Boc protection group was replaced in a known manner from this intermediate. 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 ) ⁺ ].

1.3.6

Starting from 1.3.2, the following reactions were carried out under standard conditions: Boc cleavage with trifluoroacetic acid in dichloromethane (yield: 87%), reaction with Boc-glycine-N-carboxylic acid anhydride (yield: 99%). [TLC:

R _f = 0.6 ^1} ].

1.3.7

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

The residue was then taken up again in 10 ml of dichloromethane and to a solution of 44 mg (0.24 mmol) of Boc-piperidine and 187 mg of pyridine in 10 ml

Given dichloromethane. After stirring at RT for 1 h, the solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane / methanol 97.5 / 2.5). The appropriate fractions were combined and the solvent was distilled off in vacuo. 74 mg (57%) of the fully protected intermediate [TLC: acetonitrile / water 20/1: Rf = 0.6] were obtained.

The Fmoc protective group was detached from 72 mg (0.13 mmol) of this intermediate with piperidine in DMF. According to standard conditions with EDCI / HOBT, the deprotected product was coupled with benzyloxycarbonylglycylglycine to the target product in the presence of ethyldiisopropylamine. Yield: 82%

[TLC: R _f = 0.5 ⁴⁾ ]. 1.3.8

713 mg (2.93 mmol) of N-3-aminopropyl-N'-tert-butoxycarbonyl-piperazine and 2.3 g of ethyl-diisopropylamine were placed in 100 ml of dichloromethane, and 225 mg (1.78 mmol) of oxalyl chloride were then added dropwise , After stirring at RT for 1 h, the mixture was diluted with a further 100 ml of oxalyl chloride and shaken out three times with 5% sodium hydrogen carbonate solution. The organic phase was dried over sodium sulfate and concentrated. The residue was digested with diethyl ether and the product was filtered off. The mother liquor was again with

Petroleum ether likes. 515 mg (54%) of the fully protected intermediate [TLC: R _f = 0.3 ⁶⁾ ] were obtained.

From this, the Boc protective group was replaced with trifluoroacetic acid (quantitative implementation). After standard conditions with EDCI / HOBT, the deprotected product was then coupled with Boc-glycine to the target product in the presence of ethyldiisopropylamine. Yield: 42% [TLC: R _f = 0.61 ⁹⁾ ] [ESI-MS: m / e = 655 (M + H) ⁺ ].

1.3.9

793 mg (1.38 mmol) of the compound from Example 1.3.7 were dissolved in 40 ml of methanol and 15 ml of THF and hydrogenated over palladium / activated carbon (10% Pd). After 1 h the catalyst was separated off and the solvent was evaporated off. The residue was taken up in dioxane / water and lyophilized. 512 mg (84%) of the target compound were obtained [TLC: R _f = 0.17 ⁵⁾ ].

01/03/10

49.9 mg (0.272 mmol) ω-maleimidobutyric acid and 46 mg (0.34 mmol)

1-Hydroxy-1H-benzotriazole and 52 mg (0.272 mmol) of N- (3-dimethylaminopropyl) - N'-ethylcarbodiirnide hydrochloride were combined in 20 ml of DMF and stirred at RT for 90 min. 100 mg (0.227 mmol) of the compound from Example 1.3.9 and then 120 μl of ethyl-diisopropylamine were then added. The mixture was stirred at RT for 6 h and concentrated. The residue was taken up in dichloromethane and shaken three times with water. The organic phase was separated, dried over sodium sulfate and concentrated. The residue was then precipitated from dichloromethane with diethyl ether. The precipitate was filtered off and dried in a high vacuum. 78 mg (yield: 57%) of the intermediate were obtained.

The Boc protective group was detached from this with trifluoroacetic acid in order to obtain the target compound (quantitative conversion) [TLC: Rf = 0.28 ⁶⁾ ]. 01/03/11

The production was carried out in analogy to example 1.3.10 starting from 1.3.9. Yield: 72% [TLC: R _f = 0.38 ⁶⁾ ].

01/03/12

500 mg (1.94 mmol) of N-2-carboxyethyl-N'-tert.butoxycarbonyl-piperazine were coupled with 537 mg (1.94 mmol) of benzyloxycarbonyl-glycyl-piperazine (compound E.2.6) according to standard conditions with EDCI / HOBT , 630 mg (yield: 63%) of the fully protected intermediate were obtained [TLC: R _f = 0.4 ⁵⁾ ].

400 mg of the intermediate were hydrogenated in 40 ml of methanol and over palladium / activated carbon (10% Pd). After 3 hours, the catalyst was separated off, the solvent was evaporated off and the residue was dried. 401 mg (yield: 95%) were obtained [TLC: R _f = 0.2 ⁶⁾ ].

As described in Example 1.3.10, this intermediate was coupled with ω-maleimidobutyric acid using EDCI / HOBT. Then was the Boc group was removed using trifluoroacetic acid. The target compound was obtained in a yield of 46% over 2 steps [TLC: R _f = 0.21 ⁸⁾ ].

01/03/13

The production took place in analogy to example 1.3.7 with the building blocks: Fmoc-piperidine oxalyl chloride Boc-piperidine

Connection from example E.2.9.

Yield: 52% over 4 steps [TLC: R _f = 0.4 ⁷⁾ ] [ESI-MS: m / e = 580 (M + H) ⁺ ].

01/03/14

The production took place in analogy to example 1.3.7 with the building blocks:

Fmoc-piperidine oxalyl chloride compound from Example E.2.7

Connection from example E.2.9.

Yield: 38% over 4 steps [TLC: R _f - 0.4 ⁷⁾ ] [ESI-MS: m / e = 586 (M + H) ⁺ ].

01/03/15

The production took place in analogy to example 1.3.7 with the building blocks:

Connection from example E.2.8

oxalyl

Connection from example E.2.7 Connection from example E.2.9.

Yield: 41% over 4 steps [TLC: R _f = 0.55 ⁴⁾ ] [ESI-MS: m / e = 592 (M + H) ⁺ ]. 01/03/16

The preparation was carried out in analogy to Examples 1.3.9 and 1.3.10 starting from: Compound from Example 1.3.13

Yield: 60% over 3 steps [TLC: R _f = 0.16 ⁵⁾ ]

01/03/17

The preparation was carried out in analogy to Examples 1.3.9 and 1.3.10 starting from: Compound from Example 1.3.14

Yield: 66% over 3 steps [TLC: R _f = 0.16 ⁵⁾ ] 01/03/18

The preparation was carried out in analogy to Examples 1.3.9 and 1.3.10 starting from: Compound from Example 1.3.15

Yield: 49% over 3 steps [TLC: R _f = 0.16 ⁵⁾ ]

Examples of acid-cleavable affinity markers

Example 1; Manufacturing process (variant A)

368 mg (0.63 mmol) of the intermediate 1.2.1 were taken up in 5 ml of DMF and 500 μl of piperidine were added. After stirring for 15 min at room temperature, the mixture was concentrated and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane / methanol / aqueous ammonia (17%) 15/3 / 0.3). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 204 mg (90%) of the target product were obtained [TLC: dichloromethane / methanol / ammonia 17% 15/4 / 0.5 R _f = 0.28].

26 mg (73 μmol) of this intermediate were taken up in 10 ml of DMF, and 18.4 mg (73 μmol) of maleimidoacetic acid N-hydroxysuccinimide ester and 19 mg of ethyldiisopropylamine were added and the mixture was stirred at RT for 1 h. Alternatively, maleimidoacetic acid can also be coupled with the amine component in the presence of EDCI / HOBT. The solvent was evaporated and the residue was purified by flash chromatography on silica gel (eluent ²⁾ ).

The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. 34 mg (95%) of the desired product were obtained [TLC: R _f = 0.17 ²⁾ ].

33 mg (67 μmol) of this intermediate were taken up in 5 ml of dichloromethane and 1 ml of TFA was added. After stirring for 15 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether. To Filtration and drying gave 33 mg (97%) of the desired product as the trifluoroacetic acid salt [TLC: acetonitrile / water / glacial acetic acid 10/3 / 1.5 R _f = 0.22] [ESI-MS: me = 395 (M + H ) ⁺ ].

32 mg (63 μmol) of the deprotected intermediate and 26 mg (70 μmol) of the isothiocyanate Ell from the educt series 1 were dissolved in 5 ml of DMF, mixed with 37 μl of ethyldiisopropylamine and then stirred at RT for 4 h. The mixture was concentrated, the residue was stirred with water and suction filtered. The residue was separated and then treated three times with dichloromethane / methanol 1: 1 and twice more with methanol in an ultrasound bath. After drying, 19 mg (35%>) of the target compound were obtained [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: m / e = 785 (M + H) ⁺ ].

Example 2; Manufacturing process (variant B)

182 mg (0.31 mmol) of compound 1.1.1 were taken up in 15 ml of dichloromethane and 1 ml of TFA was added. After stirring for 15 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether. After filtration and drying, 175 mg (94%) of the desired was obtained

Product as trifluoroacetic acid salt [TLC: R _f = 0.2 ⁵⁾ ].

170 mg (286 μmol) of the deprotected intermediate were placed in 10 ml of DMF and 112 mg (286 μmol) of the isothiocyanate ell from the educt series 1 and 150 μl of ethyldiisopropylamine were added and the mixture was then stirred at RT overnight. It was concentrated, the residue was stirred with 10 ml of water and suction filtered. The residue was separated and then stirred with dichloromethane. After filtration and Drying gave 244 mg (98%) of the desired compound [TLC: R _f = 0.23 ⁴⁾ ].

240 mg (0.276 mmol) of this intermediate were taken up in 10 ml of DMF and 500 μl of piperidine were added. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was digested with dichloromethane. It was then filtered off and the filter residue was suspended in a mixture of 5 ml of DMF and 10 ml of dichloromethane. After addition of 10 ml of diethyl ether, the product precipitated completely and was filtered off and dried. 135 mg (76%) of the target product were obtained [TLC: acetonitrile / water / glacial acetic acid 10/3 / 1.5 R _f = 0.2].

30 mg (46 μmol) of this intermediate and 18 mg ethyldiisopropylamine were added to a solution of 8 mg (46 μmol) maleimidopropionic acid, 9.4 mg (70 μmol) HOBT, 11 mg (56 μmol) EDCI in 10 ml DMF, the previously Pre-reacted for 30 min, added and then rubbed overnight at RT. The solvent was evaporated and the residue was stirred with 5 ml of water. The solid residue was then stirred with 5 ml of dichloromethane / methanol 1: 1 and then mixed with 5 ml of diethyl ether. The precipitated product was dried in a high vacuum. 28 mg (76%) were obtained [TLC: R _f = 0.4 ⁵⁾ ] [ESI-MS: m / e = 799 (M + H) ⁺ ].

The following examples were prepared in an analogous manner to example 1 (variant A) or example 2 (variant B). The variant is given below.

Example 3

Educts: 1.2.2, Ell; Variant A Yield: 33% over 4 stages, then conversion into the hydrochloride with 1.5 equivalents of a 0.1 M aqueous hydrochloric acid solution

R _f = 0.3 ⁶⁾ [ESI-MS: m / e = 865 (M + H) ⁺ ]

Example 4

Educts: 1.2.3, E.l.l; Variant A Special features: Instead of maleimidoacetic acid N-hydroxysuccinimide ester, maleimidopropionic acid was linked with EDCI / HOBT.

Yield: 33% over 4 stages

R _f = 0.33 ⁵⁾

[ESI-MS: m / e = 857 (M + H) ⁺ ]

Example 5

Educts: 1.2.4, E.l.l; option A

Special features: Instead of maleimidoacetic acid N-hydroxysuccinimide ester, maleimidopropionic acid was linked with EDCI / HOBT. Yield: 30% over 4 stages R _f = 0.55 ⁵⁾

[ESI-MS: m / e = 841 (M + H) ⁺ ]

Example 6

Educts: 1.2.6, E.l.l; Variant A Special features: Instead of maleimidoacetic acid N-hydroxysuccinimide ester, maleimidopropionic acid was linked with EDCI / HOBT.

Yield: 23% over 4 stages

R _f = 0.44 ⁵⁾

[MALDI-MS: m / e = 861 (M + Na) ⁺ ]

Example 7

Educts: 1.2.7, E.l.l; option A

Special features: Instead of maleimidoacetic acid N-hydroxysuccinimide ester, maleimidopropionic acid was linked with EDCI / HOBT.

Yield: 37% over 4 stages

R _f = 0.56 ⁵⁾ [ESI-MS: m / e = 841 (M + H) ⁺ ] Example 8

Educts: Ll.l, E.l.l; option A

Yield: 70% over 4 stages

R _f = 0.53 ⁵⁾

[ESI-MS: m / e = 742 (M + H) ⁺ ]

Example 9

Educts: 1.2.5, E.l.l; Variant B

Yield: 57% over 4 steps R _f = 0.28 ⁵⁾ [MALDI-MS: m / e = 878 (M + Na) ⁺ ] Example 10

Educts: 1.2.1, E.1.3; option A

Yield: 34% over 4 steps R _f -0.25 ⁵⁾ [MALDI-MS: m / e = 878 (M + Na) ⁺ ]

Example 11

Educts: 1.2.1, E.1.2; option A

Yield: 31% over 4 stages R _f = 0.4 ⁵⁾

[MALDI-MS: m / e = 807 (M + Na) ⁺ ]

Example 12

Educts: 1.1.2, Ell; option A

Yield: 34% over 4 steps R _f = 0.38 ⁴⁾ [ESI-MS: m / e = 784 (M + H) ⁺ ]

Example 13

Educts: 1.2.1, E.1.2; Variant B

Special features: Instead of the connection of maleimidopropionic acid in the presence of EDCI / HOBT, in the last step the amine precursor of the end product was reacted with ε-maleimidocaprylic acid in the presence of EDCI / HOBT.

Yield: 15 mg (19% over 4 steps)

R _f = 0.5 ⁵⁾

[ESI-MS: m / e - 827 (M + H) ⁺ ]

Example 14

Educts: 1.2.1, E.1.2; Variant B Special features: Instead of the connection of maleimidopropionic acid in the presence of EDCI / HOBT, in the last step the amine precursor of the end product was reacted with ε-maleimidobutyric acid in the presence of EDCI / HOBT.

Yield: 12% over 4 steps R _f = 0.5 ⁵⁾ [ESI-MS: m / e = 799 (M + H) ⁺ ]

Example 15

Educts: 1.2.1, E.1.2; Variant B

Special features: Instead of the connection of maleimidopropionic acid in the presence of EDCI / HOBT, the amine precursor of the end product was reacted with acrylic acid chloride (6Eq) in dichloromethane in the presence of 2 equivalents of pyridine in the last step. The resulting target compound was purified by flash chromatography (eluent: acetonitrile / water 10/1).

Yield: 3% over 4 stages

R _f = 0.15 ⁴⁾

[ESI-MS: m / e = 688 (M + H) ⁺ ] Example 16

Educts: 1.2.1, E.1.2; Variant B

Special features: Instead of the connection of maleimidopropionic acid in the presence of EDCI / HOBT, the amine precursor of the end product was reacted with chloroacetyl chloride in dichloromethane in the presence of 2 equivalents of pyridine in the last step.

Yield: 22% over 4 stages

R _f = 0.12 ^1}

[MALDI-MS: m / e = 732 (M + Na) ⁺ ]

Example 17

Educts: 1.3.4, E.1.2; Variant B

Yield: 58% over 4 stages R _f = 0.2 ⁵⁾

[ESI-MS: me = 954 (M + H) + ⁺ ] Example 18

Educts: 1.3.5, E.1.2; Variant B

Example 19

Educts: 1.3.1, E.1.2; Variant B Yield: 83% over 4 stages

R = 0.4 ⁵⁾

[ESI-MS: m / e = 925 (M + H) ⁺ ]

Example 20

Educts: 1.3.3 E.1.2; Variant B Example 21

Educts: I.2.2., E.1.2; Variant B Yield: 56% over 4 steps R _f = 0.3 ⁸⁾ [ESI-MS: m / e = 879 (M + H) ⁺ ]

Example 22

Educts: I.2.9., E.1.2; option A

Yield: 26% over 4 stages

R _f = 0.5 ⁵⁾

[ESI-MS: m / e = 871 (M + H) ⁺ ] Example 23

The preparation was carried out by converting the compound from Example 21 into the

Hydrochloride with 0.1 M aqueous HCl. R _f = 0.23 ⁶⁾ [ESI-MS: me = 879 (M + H) ⁺ ]

Example 24

Educts: Ll.l, E.l.l; Variant B

Yield: 47% over 4 stages R _f = 0.5 ⁵⁾

[ESI-MS: m / e = 756 (M + H) ⁺ ]

Example 25

Educt: E.2.3, then the following standard implementations followed: Linkage with ω-maleimidobutyric acid in the presence of EDCI / HOBT (61%), Boc elimination (88%),

Linking with bis-boc-histidine-N-hydroxysuccinimide ester (52%), Boc elimination (69%),

Implementation with E.l.l (96%).

R _f = 0.4 ⁶⁾ [ESI-MS: m / e = 836 (M + H) ⁺ ]

Example 26

Educts: 1.2.8, E.l.l; Variant B

Yield: 23% over 4 stages

R _f = 0.3 ⁵⁾

[ESI-MS: m / e = 827 (M + H) ⁺ ]

Example 27

Educts: 1.3.6, E.1.2; Variant B

Yield: 27% over 4 steps

R _f = 0.26 ⁵⁾

[ESI-MS: m / e = 911 (M + H) ⁺ ]

Example 28

Educt: 1.3.10, then the following standard implementations followed:

Linking with Boc-Glycine-N-carboxylic anhydride (71%), Boc-cleavage with trifluoroacetic acid (80%), linkage with E.l.l (54%).

R _f = 0.38 ⁵⁾ [ESI-MS: m / e = 953 (M + H) ⁺ ]

Example 29

Educt: 1.3.8, then the following standard implementations followed:

Boc cleavage at both terminal amino groups with trifluoroacetic acid (quant.), Reaction with V% Eq. Of the compound Ell to form the mono-thiourea (28%) [R _f = 0.15 ⁹⁾ ]

Reaction with ω-maleidobutyric acid in the presence of EDCI / HOBT (50%), [ESI-MS: m / e = 1010 (M + H) ⁺ ]

Example 30

Educt: 1.3.12, then the following standard implementations followed:

Reaction with Boc-Glycine-N-carboxylic anhydride (84%), Boc elimination with trifluoroacetic acid (quant.), [R _f = 0.28 ⁸⁾ ] Reaction with Ell (60%), [R _f = 0.46 ^{6 )} ] [ESI-MS: m / e = 896 (M + H) ⁺ ] Example 31

The preparation was carried out analogously to the compound from Example 30.

Implementation with E.1.2 instead of Ell (52%), [R _f = 0.55 ⁶⁾ ] [ESI-MS: m / e = 882 (M + H) ⁺ ]

Example 32

Educt: 1.3.10, then the following standard implementations followed:

Reaction with bis-boc-histidine-N-hydroxysuccinimide ester (39%)

Boc cleavage with trifluoroacetic acid (87%), [R _f = 0.44 ⁸⁾ ] reaction with class 2 (45%), [R _f = 0.44 ⁶⁾ ] [ESI-MS: m / e = 1019 (M + H) ⁺ ] Example 33

The preparation was carried out in analogy to the compound from Example 32.

Reaction with Ell instead of E.1.2 (35%), [R _f = 0.3 ⁶⁾ ] [ESI-MS: m / e = 1033 (M + H) ⁺ ]

Example 34

Educt: 1.3.11, then the following standard implementations followed:

Reaction with Boc-Glycine-N-carboxylic anhydride (60%) Boc elimination with trifluoroacetic acid (quant.), [R _f = 0.3 ⁶⁾ ] Reaction with Ell (97%), [R _f = 0.44 ⁶⁾ ] [ESI-MS: m / e = 981 (M + H) ⁺ ] Example 35

The preparation was carried out analogously to the compound from Example 34, reaction with E.1.2 instead of Ell (73%), [R _f = 0.48 ⁵⁾ ] [ESI-MS: m / e = 967 (M + H) ⁺ ]

Example 36

Educt: 1.3.11, followed by the following standard reactions: reaction with bis-Boc-histidine-N-hydroxysuccinimide ester (41%) Boc elimination with trifluoroacetic acid (quant.), [R _f = 0.12 ⁶⁾ ] reaction with Ell (90%), [R _f = 0.38 ⁶⁾ ] [ESI-MS: m / e = 1061 (M + H) ⁺ ] Example 37

The preparation was carried out in analogy to the compound from Example 36.

Implementation with E.1.2 instead of Ell (73%), [R _f = 0.4 ⁶⁾ ] [ESI-MS: m / e = 1047 (M + H) ⁺ ]

Example 38

The preparation was carried out in analogy to the compound from Example 28. Reaction with E.1.2 instead of E.1.1 (72%), [R _f = 0.45 ⁵⁾ ]

[ESI-MS: m / e = (M + H) ⁺ ]

Together with the affinity markers of Examples 39, 40 and 41, this affinity marker forms an affinity marker quadraplet which enables a parallel analysis of four protein samples. Example 39

Educt: 1.3.16, then the following standard implementations followed:

Linking with Boc-Glycine-N-carboxylic anhydride (81%), Boc cleavage with trifluoroacetic acid (98%), linkage with E.1.2 (81%) R _f = 0.5 ⁵⁾

[ESI-MS: m / e = 944 (M + H) ⁺ ]

Example 40

Educt: 1.3.17, then the following standard implementations followed:

Linking with Boc-Glycine-N-carboxylic anhydride (80%), Boc cleavage with trifluoroacetic acid (quant.),

Link with E.1.2 (74%) R _f = 0.5 ⁵⁾ [ESI-MS: me = 950 (M + H) ⁺ ] Example 41

Educt: 1.3.18, then the following standard implementations followed:

Linkage with Boc-Glycine-N-carboxylic anhydride (94%), Boc cleavage with trifluoroacetic acid (98%), linkage with E.1.2 (38%) R _f = 0.5 ⁵⁾

[ESI-MS: m / e = 956 (M + H) ⁺ ]

Example 42

Educt 1.3.10, then the following standard implementations followed:

Linking with Boc-Glycine-N-carboxylic anhydride (78%), Boc cleavage with trifluoroacetic acid (93%) [R _f = 0.2 ⁶⁾ ].

In parallel, 100 mg (0.026 mmol) of NovaSyn TG Resin 01-64-0043 were washed three times with DMF. Then 28 mg (0.08 mmol) of 4- (Fmoc-amino) benzoic acid, 30 mg of HATU, and 20 mg of diisopropylethylamine in 2 ml of DMF were added and stirred overnight at RT. It was washed four times with DMF. The Fmoc group was then removed according to standard conditions and the resin was washed four times with DMF. 2 ml of dioxane / water 1/1 and 10 μl of thiophosgene were added. After 1 h, 200 μl of ethyldiisopropylamine were added and after another 1 h the mixture was washed with water, dioxane and DMF. 35 mg of the previously prepared amine component were added in 2 ml of DMF and 25 μl of ethyldiisopropylamine. After 2 h was washed twice with DMF and THF and dried.

Example 43

Educt: E.2.10, then the following standard implementations followed:

Implementation with Z-Glu (tBu) -OSu (33%),

Hydrogenation over Pd-C (92%),

Reaction with Z-Leu in the presence of EDCI / HOBT (88%),

Hydrogenation over Pd-C (85%),

Linking with ω-maleimidobutyric acid in the presence of EDCI / HOBT (66%), Boc elimination (88%),

Implementation with E.l.l (88%).

R _f = 0.26 ⁴⁾

[ESI-MS: m / e = 941 (M + H) ⁺ ] Example 44

Educt: E.2.10, then the following standard implementations followed:

Reaction with Fmoc-Leu-Leu in the presence of EDCI / HOBT (58%), Boc elimination (78%), reaction with E.l.l (90%), Fmoc elimination with piperidine in DMF (68%),

Linkage with ω-maleimidobutyric acid in the presence of EDCI / HOBT (72%),

R _f = 0.68 ⁵⁾

[FAB-MS: m / e = 925 (M + H) ⁺ ]

Example 45

Preparation analogous to example 44; in the last step, however, the linkage takes place with ω-maleimidocaprylic acid in the presence of EDCI / HOBT (61%),

Rf = 0.4 ⁴ >

[FAB-MS: m / e = 953 (M + H) ⁺ ] Example 46

Educt: E.2.10, then the following standard implementations followed:

Reaction with Fmoc-Leu-Leu in the presence of EDCI / HOBT (58%>),

Boc release (78%),

Implementation with E.1.2 (72%),

Fmoc cleavage with piperidine in DMF (96%),

Linkage with ω-maleimidobutyric acid in the presence of EDCI / HOBT (92%),

R _f = 0.4 ⁴⁾

[ESI-MS: m / e = 911 (M + H) ⁺ ]

Example 47

Preparation analogous to example 46; in the last step, however, the linkage takes place with ω-maleimidocaprylic acid in the presence of EDCI / HOBT (61%), R _f = 0.5 ⁴⁾ [FAB-MS: m / e = 939 (M + H) ⁺ ] Example 48

Educt 1.2.1, then the following standard implementations followed:

Boc cleavage with trifluoroacetic acid (94%) [R _f = 0.2 ⁵⁾ ] This gives the amine component A.

In parallel, 42 mg (0.04 mmol) of aminopropyl silica gel (Aldrich, 36425-8, loading 0.95 nmol / g) were suspended in 2 ml of DMF and successively with 45 mg (0.12 mmol) of 4- (Fmoc- amino) -phenylacetic acid, 2 μl diisopropylethylamine, 15 mg (0.12 mmol) diisopropylcarbodiimide and 16 mg (0.12 mmol) HOBT. Allow to stand at RT overnight and then wash four times with DMF.

The Fmoc group is then removed with 2 ml of 20% piperidine in DMF and the resin is washed four times with DMF and dioxane.

Then 1 ml of dioxane and 45 μl of thiophosgene are added. After 1 h, 900 μl

Ethyldiisopropylamine was added and after another 1 hour the mixture was washed three times with dioxane, DMF and DCM.

47 mg (0.08 mmol) of the previously prepared amine component are added in 2 ml DMF and 40 μl ethyldiisopropylamine. Allow to stand at RT overnight and then wash four times with DMF.

The Fmoc grape is then removed with 2 ml of 20% piperidine in DMF and the resin is washed four times with DMF. 1 ml of DMF is added, followed by 22 mg (0.12 mmol) of 4-maleimido-butyric acid, 15 mg (0.12 mmol) of diisopropylcarbodiimide and 16 mg (0.12 mmol) of HOBT, and the mixture is stirred at RT overnight. Then it is washed three times with DMF, DCM and THF.

Protein analysis studies

Coupling of the affinity marker to SDS-7 and description of the workflow for the affinity marker from example 11

A mixture of seven proteins was used as a sample, which is also used as a size standard in gel electrophoresis (SDS-7 marker, Sigma-Aldrich GmbH, Taufkirchen).

24 μg of the protein mixture were dissolved in 5 μl buffer 1 and diluted with 135 μl buffer 3. The proteins were denatured by heating at 100 ° C. for 3 minutes. To reduce the cysteines present, 3 μl of reducing solution were added and the mixture was incubated at 100 ° C. for 10 minutes. To convert the free cysteines with the affinity marker from Example 11, 5 μl of derivatization solution was then added and incubated at 37 ° C. for 90 minutes.

After derivatization, 3 ul trypsin solution was added. The proteins are cleaved overnight (approx. 17 hours) 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 11) in DMSO trypsin solution: 1 mg / ml trypsin (Promega GmbH, Mannheim) in buffer 3 Affinity purification of derivatized peptides

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 2xPBS

- Four column volumes of 30% (v / v) acetonitrile / 0.4% (v / v) trifluoroacetic acid

- Seven column volumes of 2xPBS - Four column volumes of 2 mM biotin in 2xPBS

- Six column volumes of 100 mM glycine, pH 2.8

- Six column volumes of 2xPBS

30 ul sample was diluted with 30 ul 2xPBS 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 2xPBS

- 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

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 dissolved again shortly before the analysis with mass spectrometry.

PBS: stock solution lOx, GibcoBRL, Cat. No. 14200-067 Mass spectrometric 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 peptide with the

Sequence FLDDDLTDDIMCVK from lactalbumin, which 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.

A total of 19 different peptides from the sample were identified in an analysis, all bearing the expected mass of the affinity tag residue in the same way. Not a single cysteine-containing peptide was identified that carried a complete affinity marker.

1: fragment spectrum of a derivatized with the compound from Example 11

Peptides after isolation via avidin, i.e. H. with acid-split affinity marker.

Coupling of the Affinity Markers to Proteins and Description of the Workflow for the Affinity Markers of Examples 38 to 41 A mixture of seven proteins was used as a sample, which is also used as a size standard in gel electrophoresis (SDS-7 marker, Sigma-Aldrich GmbH, Taufkirchen). The two samples for comparison contained the following proteins in identical amounts:

Beef Seramalbumin Beef Alpha Lactalbumin Trypsin Inhibitor from Soybean Trypsin from Beef> SDS-7 Ovalbumin from Chicken

Human glyceraldehyde-3-phosphate dehoydrogenase Carbonic anhydrase J

Sample 1 was also added human interleukin-4, which was not present in sample 2.

About 240 μg of protein were used in each sample, which were distributed approximately equally over the 7 or 8 proteins mentioned above. The SDS-7 samples were first dissolved in 10 μl buffer 1 and diluted with 95 μl buffer 3. Then sample 1 was also added with 10 ul Interleuking-4 solution. The samples were

Buffer 3 filled up to 200 μl and halved in each case (samples la and lb or 2a and 2b).

The proteins were then denatured by heating at 100 ° C. for 3 minutes. To reduce the cysteines present, 3 μl of reducing solution were added and the batches were incubated at 100 ° C. for 10 minutes. To convert the free cysteines with the affinity markers from the examples, 5 μl of derivatization solution were added to each sample and incubated at 37 ° C. for 120 minutes. Then all 4 samples were mixed and half of the total sample was processed. Sample la: reaction with example 38 sample lb: reaction with example 41 sample 2a: reaction with example 39 sample 2b: reaction with example 40

After the reaction, the proteins were precipitated by adding four times the volume of ice-cold acetone / ethanol 1: 1 for 15 minutes at -20 ° C. The sediment was washed once with acetone ethanol / water 4: 4: 2 and then dried in vacuo. The sample was dissolved in 10 μl buffer 1 and diluted with 260 μl buffer 3. 40 μl of trypsin solution were added to cleave the proteins and the sample was incubated at 37 ° C. for 1 h. The sample was then briefly heated to 95 ° C. to inactivate the enzyme.

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: 36 µg / µl affinity marker (Examples 38-41) in DMSO

Trypsin solution: 1 mg / ml trypsin (Promega GmbH, Mannheim) in buffer 3

Affinity purification of derivatized peptides

For the selective purification of the derivatized peptides from the sample, an affinity purification was carried out using a self-made avidin column (Monomeric Avidin, Perbio Science Deutschland GmbH, Bonn). The column was manufactured and used in accordance with the manufacturer's instructions. The peptides were eluted with 0.4% trifluoroacetic acid / 30% acetonitrile.

Mass spectrometric analysis

An ion trap mass spectrometer (LCQdeka, ThermoFinnigan, San Jose) was used to analyze the peptides. time chromatography is connected (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.

Based on the experimental approach, the following results were expected:

• All peptides from the SDS-7 mixture should be detected in the form of four identically intense signals, which show no isotope effect in the chromatography.

• Interleukin-4 peptides should be in the form of a doublet of signals with a

Mass difference of 17 da occur.

2 shows the ion traces for the four differently labeled variants of the peptide LQGrVSWGSGCAQK from trypsinogen. From top to bottom, it is the peptide marked with the affinity markers with 0, 5, 11 and

17 isotope labels. A retention time difference between the variants cannot be measured. 3 shows the associated MS spectrum, which contains a quadraplet of approximately equally intense signals. The identity of the peptide was confirmed by MS / MS experiments.

4 shows the signals of the peptide NLWGLAGLNSCPVK from Interleukin-4. As expected, a doublet of signals with an intensity ratio of 1: 1 appears. In this way, the signal can be clearly distinguished from peptides that are unlabeled and that have been purified by non-specific adsorption on the affinity column. The identity of the peptide was confirmed by MS / MS experiments. Fig. 2: The ion traces for the m / z values 647.3 Da, 648.9 Da, 650.9 Da and 652.9 Da. It is the triple-charged ion of the peptide LQGIVSWGSGCAQK in the forms reacted with Examples 38 to 41. The identity of the peptides was confirmed by MS / MS experiments.

3: MS spectrum under the LC peaks shown in FIG. 2. It is a triple charged peptide ion.

Fig. 4: MS spectrum of the triple-charged ion of the peptide NLWGLAGLNSCPVK from Interleukin-4. Since it was only present in Sample 1, a doublet of signals occurs, corresponding to the peptide with 0 or 17 isotope marker orange.

Claims

claims

1. Organic compound of the formula (I),

A-L-PRG (I)

in the

A for an affinity ligand residue or for a solid phase,

PRG for a protein reactive group and

L stands for an A and PRG covalently linking linker,

characterized in that the linker L contains a group of the formula (V),

in the

the glycine residue is NH-CH ₂ -CO,

L 'is a bridge that forms a covalent link between two piperazine

Residues enabled or facilitated

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, Z 'is the glycine residue CO-CH ₂ -NH, which differs from Z in the different orientation, and

k, 1, m, n each independently represent a number from 0 to 10, the sum k + 1 + m + n being at least 1 and at most 40,

or their salt.

2. Connection according to claim 1, characterized in that the linker L is cleavable.

3. A compound according to claim 2, characterized in that the linker L has a chemically, photochemically, enzymatically or thermally cleavable functional group, in particular an acid cleavable group.

4. A compound according to claim 3, characterized in that the linker L is an acid-cleavable group S of the formula

in the

Y for a spacer with 1 to 10 non-hydrogen atoms, which enables or facilitates the connection of the aryl radical to A, and

SK stands for the rest of an amino acid side chain in the D or L or in the racemic form. Compound according to claim 4, characterized in that the acid-cleavable group S connects Grappe A with the Grappe of formula (T).

Compound according to one of the preceding claims, characterized in that the linker L consists of the acid-cleavable Grappe S and the Grappe of the formula (T).

Compound according to one of the preceding claims, characterized in that the protein-reactive Grappe PRG is a selective reactivity for a terminal functionality of an amino acid, for a phosphate group or for a possibly previously generated aldehyde or keto-Grappe in the protein or for the reaction product of a targeted implementation of the Has protein.

Compound according to one of the preceding claims, characterized in that the protein-reactive Grappe -PRG is selected from

CO- [CH ₂ ] _r -Cl with r = 1-10 and

CO-C H = CH ₂ .

Compound according to one of the preceding claims, characterized in that A is an affinity ligand residue, preferably the residue of biotin, a biotin derivative, a carbonate, a hapten or a complexing agent, in particular biotin or a biotin derivative.

10. A compound according to any one of claims 1 to 8, characterized in that A is a solid phase, preferably a modified natural or synthetic resin with functional elements suitable for linking the linker L.

Groups, in particular an amino-functionalized resin based on polyethylene glycol or silica gel.

11. A method for producing a connection according to spoke 1, in which

i) a protected intermediate of the formula (III),

where SG and SG 'represent two orthogonal protecting groups,

will be produced,

ii) the protective group SG is first detached from the intermediate of the formula (III) and then a further amino acid derivative which has an identical or different protective group SG to the α-amino function which is detached, is attached, a derivative of the formula (IV) .

SG-NH-CH (SK) -CO- (Z) _k (Z ') _n -SG' (IN) in which SK stands for the side chain of an amino acid,

be obtained

iii) after detachment of the protective group SG 'from the derivative of the formula (IV) with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula (V),

U-PRG (V)

in which U stands for a group that enables PRG to be linked to Z 'or possibly to another end group of L,

is implemented

iv) the terminal protective group SG is removed, a conjugate of the formula (VI)

H ₂ N-CH (SK) -CO- (Z) _k (VI)

will get

v) an affinity ligand A-OH or A-NH ₂ or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH or A-NH ₂ or an activated form thereof with a compound of formula (VII),

in which Y stands for the optionally branched spacer group,

which can optionally carry a protective group to the derivative of the formula (VIII)

is implemented

vi) the derivative of the formula (VIII) is then converted into a corresponding isothiocyanate after prior splitting off of an optionally introduced protective group,

vii) the isothiocyanate is then coupled with the conjugate of the formula (VI) to the thiourea of the formula (IX) and

vii) in an optional last step, any protective groups still present are split off,

the successive steps v) and vi) can be carried out at any time before step vii).

12. A method for producing a compound according to claim 1, wherein

i) a protected intermediate of the formula (III),

where SG and SG 'represent two orthogonal protecting groups,

will be produced,

ii) the protective group SG is first removed from the intermediate of formula (III) and then a further amino acid derivative which has an identical or different protective group SG to the α-amino function is attached, a derivative of the formula (IV) .

SG-NH-CH (SK) -CO- (Z) _k (IV)

in which SK stands for the side chain of an amino acid,

be obtained

iii) the terminal protective group SG is detached, a conjugate of the formula (VT)

H ₂ N-CH (SK) -CO- (Z) _k -N N-L '' N IM- (Z ') _n -SG' (VI ') m

will get iv) an affinity ligand A-OH or A-NH ₂ or a hydroxyl-, carboxy- or amino-functionalized solid phase A-OH or A-NH ₂ or an activated form thereof with a compound of formula (VII),

in which Y stands for the optionally branched spacer group,

which can optionally carry a protective group, to the derivative of the formula (VIII)

is implemented

v) the derivative of the formula (VIII) is then converted into a corresponding isothiocyanate after prior splitting off of an optionally introduced protective group,

vi) the isothiocyanate is then coupled with the conjugate of the formula (VP) to form the thiourea of the formula (X ') and

vii) after detachment of the protective group SG 'from the thiourea of the formula (X') with the derivative of a protein-reactive group or the activated precursor of the derivative of a protein-reactive group of the formula (V),

U-PRG (V)

in which U stands for a group which enables PRG to be linked to Z 'or, if appropriate, to another end group of L,

is implemented and

viii) in an optional last step, any protective groups still present are split off,

wherein the successive steps iv) and v) to any

Time before step vi) can be carried out.

13. Use of one or more differently isotope-labeled compounds according to one of claims 1 to 9 as a reagent for mass spectrometric analysis of proteins.

14. Use according to claim 13 for the identification of one or more proteins or protein functions in one or more protein-containing samples.

15. Use according to claim 13 for determining the relative expression levels of one or more proteins in one or more protein-containing samples.

16. Kit for mass spectrometric analysis of proteins, containing as

Reagents one or more differently isotope-labeled compounds according to one of claims 1 to 10.