DE10234416A1 - Isotope-coded affinity markers 2 - Google Patents

Abstract

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

Description

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

Proteomics technology opens up the possibility of analyzing biological systems at the protein level to new biological targets and markers identify. It is known that of the proteins encoded in the genome only a certain part of all possible proteins is expressed, where for example tissue type, stage of development, activation of receptors or cellular interactions that affect expression patterns and rates. Around Differences in the expression of proteins in healthy or diseased tissue Determine different comparative methods for analyzing Protein expression patterns are used ((a) S.P. Gygi et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 9390; (b) D.R. Goodlett et al., Proteome Protein Anal., 2000, 3; (c) S.P. Gygi et al., Curr. Opin. Biotechnol., 2000, 11, 396).

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

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

The object of the present invention was to provide improved Affinity tag.

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

AL-PRG (I)

in the
A for an affinity ligand residue,
PRG for a protein reactive group and
L stands for an A and PRG covalently linking linker,
wherein the linker L is an acid-cleavable group S of the formula


contains in the
Y for an optionally branched spacer group with a chain length of 1 to 10, preferably 1 to 5 non-hydrogen atoms and
SK stands for the side chain of an amino acid,
or their salts.

The invention furthermore relates to the use of one or more Different isotope-labeled compounds according to the invention as a reagent for mass spectrometric analysis of proteins, especially for identification of one or more proteins or protein functions in one or more Protein-containing samples and to determine the relative expression levels of one or more proteins in one or more protein-containing samples.

The acid-cleavable group 5 ensures as a predetermined breaking point under the influence of acid a cleavage of the affinity marker, so z. B. the release of the To facilitate affinity column to reduce the remainder of the peptide and / or to make the work processes more efficient overall. This acid labile The predetermined breaking point enables the peptide fragments to be decomplexed by a much more efficient streptavidin-based affinity chromatography for Biotin-modified peptide fragments. It is also advantageous; that after acid splitting to the The remaining peptide fragments have a significantly lower molecular weight and have a higher isotope density. Furthermore, the handling of the Affinity markers through better solubility and through a crystalline or amorphous Appearance improved over the prior art.

The spacer group Y contained in the acid-cleavable group 5 is bonded to the benzene ring in the ortho, meta or para position to the nitrogen atom, the para position being preferred. Suitable spacer groups Y are, for example, chains composed of the building blocks NH, CH ₂ and / or CO, in which one or more hydrogen atoms can be substituted by identical or different, optionally heteroatom-containing hydrocarbon radicals, in particular C ₁ -C ₄ -alkyl radicals , Y preferably contains at least one NH group, in particular at its end facing away from the benzene ring. Particularly preferred spacer groups Y are NH, NH-CH ₂ and NH-CH ₂ -CH ₂ -NH-CO, the latter two preferably being bonded to the benzene ring with the CH ₂ or CO group.

The amino acid side chain SK is the side chain of an α-amino acid of the formula SK-CH (NH ₂ ) -COOH, which may be present in the D, L or racemic form for other SK than an H atom. Suitable SK are, for example, the side chains of the 20 natural amino acids and their D-forms and racemates, e.g. B. the side chains of L-glycine, L-histidine, L-valine, D-valine, L-proline, L-asparagine, L-aspartic acid and L-glutamic acid. Any other functional groups present in SK, for example in the case of amino acids such as histidine or aspartic acid, can optionally be present free or protected with a protective group.

The affinity ligand A serves for the selective enrichment of samples with the aid of the Affinity chromatography. The affinity columns are with the corresponding ones complementary partners to the affinity ligands, which are covalent or enter into non-covalent bonds with the affinity ligands. An example for a suitable affinity ligand is biotin or a biotin derivative that matches the complementary peptides avidin or streptavidin strong, non-covalent Binds. In this way, samples to be examined can be analyzed using the Affinity chromatography can be selectively isolated from sample mixtures. in the For example, carbohydrate residues that are not covalent can also have the same meaning Can interact with, for example, fixed lectins, than Affinity ligand can be used. Furthermore, in the same sense Interaction of haptens with antibodies or the interaction of Transition metals with corresponding ligands are used as complexing agents or other systems that interact with each other. In yet In a further embodiment, the affinity ligand A can be functional Represent group that a covalent fixation of the affinity marker reagent enables a polymer matrix.

In a preferred embodiment of the compounds according to the invention A for the acyl residue of an affinity ligand, for example biotinyl or a biotin Derivative.

Protein-reactive groups PRG serve for the targeted labeling of the proteins selected functional groups. PRGs have a specific reactivity for terminal functional groups of the proteins. Amino acids as elements of Proteins that are commonly used for targeted labeling for example mercaptoaminomonocarboxylic acids such as cysteine, Diaminomonocarboxylic acids such as lysine or arginine or monoaminodicarboxylic acids such as Aspartic acid or glutamic acid. Furthermore, protein-reactive groups also phosphate-reactive groups such as metal chelates and aldehyde-reactive and ketone-reactive groups such as amines Sodium borohydride or sodium cyanoborohydride. Groups can do the same be after a targeted protein derivatization such as one Bromocyan digestion or an elimination of phosphate groups etc. with the Implementation products react.

In a preferred embodiment of the compounds according to the invention, PRG stands for the remainder 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 the linker L, preferably bridged electrophiles such as


or another known protein reactive group, as z. B. by WH Scouten in Methods in Enzymology, Volume 135, edited by Klaus Mosbach, AcademicPress Inc. 1987, p. 30ff.

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, trifluoroacetates or alkali metal salts and be used.

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

Preferred compounds according to the invention are those of the formula (II)

ASB ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ (CH ₂ ) _p -X ⁴ -PRG (II)

in which
A represents the acyl residue of an affinity ligand, for example biotinyl or a biotin derivative,
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 X ⁴ , preferably bridged electrophiles such as


or another known protein reactive group, as z. B. from WH Scouten in Methods in Enzymology, Volume 135, edited by Klaus Mosbach, AcademicPress Inc. 1987, p. 30ff.
S is the acid-cleavable group according to the invention,
X ¹ , X ² , X ³ independently of one another and also for X ² independently of other X ² each for O, S, NH, NR, CO, CO-O, O-CO, CO-S, S-CO, SS, SO, SO ₂ , CO-NR, NR-CO, CS-NR, NR-CS, Si-O, O-Si, arylene or diarylene groups, wherein X ¹ , X ² , or X ^{3 are also} partially or completely not can be present
X ^{4 represents} O, S, NH, NR, CO-NR, NR-CO, CS-NR, NR-CS, arylene or diarylene groups, particularly preferably NH, NR, O or S,
m, n, p, q, r, z, s, x each independently represent a number from 0 to 100, the sum n + xm + p preferably being less than 100 and particularly preferably between 5 and 30,
B ^{1 stands} for an optionally present amine group NRR 'with the connectivity S-NRR'-X ¹ , which links as a bridge S with X ¹ , where B ¹ together with X ^{1 can be} part of an amino acid derivative, and
R, R 'independently of one another each represent hydrogen, alkyl, alkenyl, alkynyl, alkoxy or aryl or both together with N represent an N-heterocycle NRR', where R 'is additionally bonded to X ¹ and therefore cannot stand for hydrogen, where R and R 'in addition to X ^{1 can} be further modified by functional groups which, for example, improve solubility, such as. B. carboxyl or amino groups, and wherein R and R 'may be substituted in the form that B ¹ -X ¹ together give the derivative of an amino acid, and wherein the functional groups not involved in binding with S or with CH ₂ , for example contribute to improved solubility.

In the context of the present invention, alkyl, alkenyl, alkynyl, alkoxy, Aryl, arylene and N-heterocyclyl, unless otherwise specified, the following Importance:

Alkyl per se and "alk" in 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.

Alkenyl stands for an alkyl radical with at least 2 carbon atoms and in Rule 1, 2 or 3 double bonds, for example and preferably for ethenyl, n- Propenyl and methylethenyl.

Alkynyl represents an alkyl radical with at least 2 carbon atoms and in Rule 1, 2 or 3 triple bonds, for example and preferably for ethynyl and propinyl.

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

Aryl is a mono- to tricyclic aromatic, carbocyclic radical usually 6 to 14 carbon atoms, by way of example and preferably for phenyl, Naphthyl and phenanthrenyl. Arylene represents a bivalent aryl radical, for example and preferably for phenylene, naphthylene and phenanthrenylene.

N-heterocyclyl stands for a mono- or polycyclic, preferably mono- or bicyclic, non-aromatic heterocyclic radical with generally 4 to 10, preferably 5 to 8 ring atoms and at least one N-hetero atom and a total of up to 3, preferably up to 2 heteroatoms and / or hetero groups from the series N, O, S, SO, SO ₂ . The N-heterocyclyl residues can be saturated or partially unsaturated. Preferred are 5- to 8-membered, monocyclic saturated N-heterocyclyl radicals with a total of up to two heteroatoms from the series O, N and S, such as, for example and preferably, pyrrolidin-2-yl, pyrrolidin-3-yl, pyrrolinyl, piperidinyl, morpholinyl and perhydroazepinyl, especially pyrrolidin-2-yl and piperidin-4-yl.

The non-isotope-labeled compounds already represent isotope coding. For further isotope coding, the compounds according to the invention are preferably isotope-labeled with at least one carbon atom of the ¹³ C isotope, in particular four to 15 ¹³ C atoms. The disadvantageous isotope effect in the LC observed with ¹ H / ² D affinity markers is significantly reduced by the ¹² C / ¹³ C isotope coding. Alternatively or additionally, the isotopes ² D, ¹⁵ N, ¹⁷ O, ¹⁸ O and / or ³⁴ S can also be used for labeling.

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

The isotope labeling is usually carried out in L and / or PRG, in particular in L and in the case of compounds of the formula (II) in CH ₂ groups, B ¹ and / or X ¹ .

The acid-cleavable group S is preferably at the PRG-terminal end of the linker L. arranged and the spacer group Y directly to the protein-reactive group PRG bound, for example in the form of compounds of formula (II).

The degree of acid cleavage of the acid cleavable group 5 depends on the structure of the respective linker L and can be modulated and thus adapted to the requirements of the particular use of the compounds. If, for example, acid cleavage is desired under mild conditions, compounds of the formula (II) in which B ^{1 represents} an amine group NRR 'in which R is not hydrogen, in which R and R' in particular both represent alkyl or, together with N, represent N-heterocyclyl, for example and preferably the amine groups N (CH ₃ ) CH ₂ , pyrrolidin-2-yl and piperidin-4-yl. Such mild conditions are given, for example, in dilute trifluoroacetic acid, e.g. B. when diluted to a content of less than 50 vol .-%, especially less than 20 vol .-%, in a mixture of acetonitrile and water in a volume ratio of 1 to 1

The compounds of the invention can be prepared, for example, by a group

HB ¹ -X ¹ (CH ₂ ) _n - [X ² (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (III)

with V = H or OH, preferably H,
is first reacted with an amino acid derivative protected on the N-terminal amino group according to coupling methods common in peptide chemistry to give a conjugate

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (IV )

with SG a protective group common in peptide chemistry, preferably Boc and with SK the rest of an amino acid side chain in the D or L configuration or in the racemic form.

Then (IV) with the derivative of a protein-reactive group or the activated precursor of a protein-reactive group of the formula

U-PRG (V)

with U a group which enables PRG to be linked to X ⁴ , for example by becoming a leaving group together with the rest V in (IV),
be implemented. 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 amino protective group SG is then removed, a conjugate of the formula (VI) being obtained

H ₂ NCH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -PRG (VI)

In parallel, the affinity ligand A-OH or an activated form thereof, for example an activated ester or an acid chloride, is combined with a compound under suitable coupling conditions


which can optionally also carry a protective group for connection


implemented. The compound (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).

In the last step, any protective groups still present can optionally be split off in order to obtain conjugates of the formula (II).

ASB ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -PRG (II)

In a preferred embodiment of the manufacturing process, (IV) comprises a compound of the formula

Boc-NH-CH (SK) -CO-NH- (CH ₂ ) ₃ -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₃ -NH ₂ (X)

that reacts with a compound of the formulas (XIa-c)


and after detachment of the Boc protective group in the presence of ethyldiisopropylamine or another suitable base with an isothiocyanate generated from compound (VIII), for example the isothiocyanate


to the target compounds of formula (II) is implemented.

In all reaction steps, reversibly removable protective groups, as in peptide chemistry are used. A protection group SG can be preserved or simultaneously with the Boc protecting group or in a separate one Step to be replaced. Suitable protective groups are, for example, those with Trifluoroacetic acid cleavable Boc protective group or that with piperidine or Morpholine cleavable Fmoc protecting group. Other suitable protecting groups and the Appropriate methods of introduction and spin-off have been B. described in (a) Jakubke / Jeschkeit: amino acids, peptides, proteins; Verlag Chemie 1982 or (b) 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 according to the invention can also be built up in reverse order, initially in a derivative of the formula

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (IV )

a suitable further protective group SG ', preferably the Fmoc protective group, by standard methods ((a) Jakubke / Jeschkeit: amino acids, peptides, proteins; Verlag Chemie 1982; (b) Houben-Weyl, methods of organic chemistry, Georg Thieme Verlag Stuttgart, Fourth edition; Volume 15.1 and 15.2, edited by E. Wünsch) is introduced, whereby

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -SG '( XIII)

arises.

In the next step, the N-terminal protective group SG is removed and the derivative (XIII) obtained is then reacted with the isothiocyanate generated from (VIII), such as (XII), to give compounds of the formula

ASB ¹ -X ¹ (CH ₂ ) _n - [X ² (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -SG '(XIV).

After the protective group SG 'has been detached, for example the Fmoc protective group with piperidine, the coupling with the derivative of a protein-reactive group or the activated precursor of a protein-reactive group is carried out in the last step

U-PRG (V)

made and the conjugate

ASB ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -PRG (II)

receive.

The reactions can be carried out at various pressure and temperature ratios, usually at 0.5 to 2 bar, preferably under normal pressure, ie at about 1 bar, and -30 to + 100 ° C, preferably -10 to + 80 ° C, in particular 0 to 30 ° C. It is carried out in suitable solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloroform, C ₁ -C ₄ alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned. As a rule, reactions in DMF, dichloromethane, THF, dioxane / water or THF / dichloromethane at room temperature (RT), ie about 20 ° C., or under ice cooling and normal pressure are preferred.

Examples

used abbreviations

Boc: tert-butoxycarbonyl

DIEA: Diisopropylethylamine (Hünig's base)

DMAP: dimethylaminopyridine

DMF: dimethylformamide

DMSO: dimethyl sulfoxide

EDCI: N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide, x HCl

EI: electron impact ionization

ESI: electrospray ionization

Fmoc: fluorenyl-9-methoxycarbonyl

HOBT: 1-hydroxy-1H-benzotriazole

HPLC: high-performance liquid chromatography

MALDI: Matrix-assisted laser desorption ionization

MS: mass spectroscopy

MTBE: methyl tert-butyl ether

RP: Reverse phase

RT: room temperature

TCEP: triscarboxyethylphosphine

TFA: trifluoroacetic acid

THF: tetrahydrofuran

TLC: Thin layer chromatography

(v / v): Concentration in volume per volume

(w / v): concentration in mass per volume

watery: watery

The specification of the composition of solvent and eluent mixtures Unless expressly stated otherwise, this is done by separating them from "/" Specification of the components and adjustment of the relative volume parts. So means z. B. "Acetonitrile / water 10/1" a mixture of acetonitrile and water in Volume ratio of 10 to 1.

Preferred eluents (reference by specifying ¹⁾ etc.):
¹⁾ Acetonitrile / water 10/1
²⁾ Acetonitrile / water 20/1
³⁾ dichloromethane / methanol 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

For the production of exemplary affinity markers described below, the biotin derivatives of the educt series 1 and the intermediates of the educt series 2 were first produced. These starting materials were then converted to the corresponding affinity markers. Educt series 1 biotin derivatives E.1.1

1 g (4.09 mmol) biotin, 500 mg (4.09 mmol) 4-aminobenzylamine and 830 mg (6.14 mmol) 1-hydroxy-1H-benzotriazole, 942 mg (4.91 mmol) N- (3rd -Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride and 1587 mg ethyl-diisopropylamine were combined in 40 ml DMF. The mixture was stirred at room temperature overnight and concentrated and the residue was 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 was 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.1.1 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 these z. B. in Houben Weyl; Methods of organic chemistry; Fourth edition; tape 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) biotin were taken up in 10 ml dichloromethane and with 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 S00 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 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 [TLC: R _f = 0.68 ²⁾ ] were obtained.

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 removed and the residue dried in vacuo. 147 mg (82%) of the deprotected intermediate were obtained [TLC: acetonitrile / water / glacial acetic acid 5/1 / 0.2: R _f = 0.18].

191 mg (0.78 mmol) of biotin were taken up in 10 ml of DMF and with 158 mg (1.17 mmol) of 1-hydroxy-1H-benzotriazole and 180 mg (0.94 mmol) of N- (3-dimethylaminopropyl) -N '-ethylcarbodümid hydrochloride added. 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 intestine was stirred again at RT for 6 h, concentrated and the crude product was precipitated from dichloromethane with diethyl ether. The residue was separated 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 target product was precipitated from dichloromethane / methanol with diethyl ether. Yield: 230 mg (95%) [TLC: acetonitrile / water / glacial acetic acid 5/1 / 0.2: R _f = 0.44] [ESI-MS: m / e = 448 (M + H) ⁺ ]. Educt series 2 E.2.1

To a solution of 4,7,10-trioxa-1,13-tridecanediamine (1 g, 4.54 mmol) in 30 ml dichloromethane was added 913.2 mg (4.54 mmol) Boc-glycine-N-carboxy-anhydride and 100 mg of 4-dimethylaminopyridine were added and the mixture was stirred at RT for 16 h. The mixture was then concentrated under reduced pressure, the residue was taken up in dichloromethane and washed with a little water. The organic phase was then dried over sodium sulfate and concentrated, and the residue was purified by flash chromatography (eluent: dichloromethane / methanol / aqueous ammonia (17%) 15/3 / 0.3). The appropriate fractions were collected, the solvent was evaporated off and the residue was dried under high vacuum. 333 mg (20%) of an oil were obtained [TLC: R _f = 0.26 ⁵⁾ ]. E.2.2

To a solution of 4,7,10-trioxa-1,13-tridecanediamine (195 mg, 0.88 mmol) in 20 ml dichloromethane were added 400 mg (0.88 mmol) bis-tert-butoxycarbonyl-histidine-Nhydroxysuccinimideter and Stirred for 30 min at RT. The solvent was then removed in vacuo and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane / methanol / ammonia 17% 15/3 / 0.3). The appropriate fractions were combined, the solvent evaporated in vacuo. The residue was taken up in dichloromethane and shaken out with water. The organic phase was dried over sodium sulfate and then concentrated. 165 mg (34%) of an oil were obtained [TLC: R _f = 0.31 ⁵⁾ ]. E.2.3

500 mg (2.3 mmol) of Boc-D-valine as well as 467 mg (3.45 mmol) of 1-hydroxy-1Hbenzotriazole and 530 mg (2.76 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride combined in 25 ml of DMF and stirred at RT for 30 min. 507 mg (2.3 mmol) of 4,7,10-trioxa-1,13-tridecanediamine were then added and the mixture was stirred at RT overnight. The mixture was then concentrated, the residue was taken up in dichloromethane and extracted twice with water. The organic phase was then concentrated and the residue was purified by flash chromatography on silica gel (eluent ⁷⁾ ). The appropriate fractions were combined and the solvent was evaporated in vacuo. The residue was taken up in dichloromethane and shaken out with water. The organic phase was dried over sodium sulfate and then concentrated. 220 mg (23%) of an oil were obtained [TLC: R _f = 0.29 ⁵⁾ ]. E.2.4

A solution of chloroformic acid (9-) was added to the solution of 4,7,10-trioxa-1,13-tridecanediamine (20.0 g; 90.8 mmol) in 2-propanol (2000 ml) at -10 ° C. fluorenylmethyl) ester (23.5 g; 90.8 mmol) in tetrahydrofuran (500 ml) was slowly added dropwise. After 2 h, the mixture was concentrated under reduced pressure. The residue was taken up in dichloromethane (1000 ml) and washed twice with saturated sodium chloride solution (250 ml each). The organic phase was dried and concentrated. The residue was largely uniform and free of the starting product 4,7,10-trioxa-1,13-tridecanediamine. The yield of crude product was 36.5 g (91%) in the form of a syrup [TLC: dichloromethane / methanol 5/1: R _f = 0.27] [ESI-MS: m / z 443.4 (M + H) ⁺ ]. E.2.5

378 mg (2.2 mmol) of Boc-Glycine as well as 364 mg (2.7 mmol) of 1-hydroxy-1H-benzotriazole and 413 mg (2.16 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride combined in 30 ml of DMF and stirred at RT for 30 min. Then 1000 mg (1.8 mmol) of compound E.2.4 were added and the mixture was stirred at RT for 2 h. The mixture was then concentrated, the residue was taken up in dichloromethane and extracted twice with water. The organic phase was then concentrated and the residue was purified by flash chromatography on silica gel (eluent 2). The appropriate fractions were combined and the solvent was evaporated in vacuo. The residue was taken up in dichloromethane and shaken out with water. The organic phase was dried over sodium sulfate and then concentrated. 797 mg (76%) of an oil were obtained [TLC: R _f = 0.64 ¹⁾ ]. E.2.6

The Boc protective group became the 790 mg (1.35 mmol) of the compound E.2.5 detached with trifluoroacetic acid in dichloromethane according to standard conditions (800 mg, 98%).

790 mg (1.32 mmol) of the deprotected intermediate was taken up in 30 ml of DMF and 514 mg (1.32 mmol) of the compound E.1.1 and 511 mg of N, N-diisopropylethylamine were added and the mixture was stirred at RT for 16 h. The solvent was evaporated, the residue was stirred with 30 ml of water and the remaining solid was filtered off. It was taken up in dichloromethane / methanol and then the target compound was precipitated with diethyl ether. After filtration and drying, 1077 mg (93%) were obtained [TLC: R _f = 0.4 ⁴⁾ ].

1070 mg (1.22 mmol) of this intermediate were taken up in 25 ml of DMF and 1 ml of piperidine was added. After stirring for 45 min at room temperature, the mixture was concentrated and the residue was digested with dichloromethane. The mixture was then filtered off and the filter residue was suspended in a mixture of 10 ml of dichloromethane and 10 ml of methanol. After addition of 100 ml of diethyl ether, the product precipitated completely and was filtered off and dried. 805 mg (99%) of the target product E.2.6 [TLC: acetonitrile / water / glacial acetic acid 10/3 / 1.5 R _f = 0.4] were obtained. E.2.7

Diethylene glycol (3.18 g; 30 mmol) was added dropwise to a solution of aqueous potassium hydroxide (40%; 120 mg) and 1,4-dioxane (3.75 ml). Acrylonitrile-1,2,3- ( ¹³ C) ₃ (3.36 g; 60 mmol) was added dropwise to the solution while cooling with ice. After warming to room temperature, stirring was continued for 16 h. The solution was diluted with dichloromethane (30 ml) and washed twice with saturated saline. The organic phase was dried and concentrated. The residue was purified by column chromatography (eluent: dichloromethane / methanol 50/1). Yield: 6.21 g (95%), consistency: syrup [TLC: dichloromethane / methanol 10/1: R _f = 0.71; MS (ESI): m / z 241.1 (M + Na) ⁺ , 219.1 (M + H) ⁺ ]. E.2.8

Raney nickel (2.5 g) was added to the solution of compound E.2.7 (5.0 g; 22.9 mmol) in methanol (115 ml) and concentrated aqueous ammonia solution (68 ml) and for 5 h at 100 ° C and 100 bar hydrogenated with hydrogen. After cooling to room temperature, the catalyst was suctioned off. The filtrate was concentrated. The residue was taken up three times in ethanol and concentrated. Yield: 3.84 g (74%), consistency: syrup [TLC: dichloromethane / methanol / ammonia 4/3/1: R _f = 0.27] [MS (ESI): m / z 227.3 (M + H) ⁺ ; ¹³ C NMR (100.6 MHz, CDCl ₃ ): δ = 69.48, 69.10 ( ¹³ C H ₂ -O), 39.13, 38.77 ( ¹³ C H ₂ -NH ₂ ), 32 , 74, 32.38, 32.36, 32.01 ( ¹³ CH ₂ - ¹³ C H _2- 13CH ₂ )]. E.2.9

A solution of chloroformic acid (9-fluorenylmethyl) ester (1.14 g.) Was added to the solution of compound E.2.8 (1.0 g; 4.42 mmol) in 2-propanol (50 ml) at -10.degree ; 4.42 mmol) in tetrahydrofuran (20 ml). It was slowly warmed to room temperature and stirred for 16 h. The mixture was concentrated. The residue was taken up in dichloromethane (50 ml), washed twice with saturated aqueous sodium chloride solution, dried and concentrated. The residue is largely uniform and was used in the next stage without further purification. Yield: 1.76 g (89%); Consistency: syrup [TLC: dichloromethane / methanol 5/1: R _f 0.27] [MS (ESI): m / z 449.4 (M + H) ⁺ ]. E.2.10

The Boc protective group was introduced into 15 g (68 mmol) of 4,7,10-trioxa-1,13-tridecanediamine under standard conditions using di-tert-butyl dicarbonate in dichloromethane [TLC: R _f = 0.34 ⁵⁾ ]. E.2.11

372 mg (2 mmol) maleimidobutyric acid as well as 316 mg (2.34 mmol) 1-hydroxy-1Hbenzotriazol and 359 mg (1.87 mmol) N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride were combined in 25 ml DMF and Stirred at RT for 1 h. S00 mg (1.56 mmol) of the compound E.2.10 and 403 mg of ethyldiisopropylamine were then added and the mixture was stirred at RT for a further 16 h. The mixture was then concentrated, the residue was taken up in dichloromethane and extracted four times with water. The organic phase was then dried over sodium sulfate and concentrated, and the residue was precipitated from dichloromethane using petroleum ether. The supernatant was decanted off and the remaining oily residue was dried under high vacuum. 577 mg (76%) of the desired product were obtained [TLC: R _f = 0.48 ⁴⁾ ].

570 mg (1.17 mmol) of this intermediate were taken up in 10 ml dichloromethane and 1 ml TFA was added. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether. After filtration and drying, 90 mg (85%) of the desired product were obtained as the trifluoroacetic acid salt [TLC: R _f = 0.2 ⁵⁾ ]. E.2.12

Production in analogy to Example E.2.10 and E.2.11 starting from Example E.2.8 [TLC: R _f = 0.1 ⁵⁾ ]. E.2.13

Production in analogy to Example E.2.10 and E.2.11 starting from Example E.2.8 [TLC: R _f = 0.2 ⁵⁾ ]. E.2.14

125 mg (0.4 mmol) of tert-butoxycarbonyl-proline-N-hydroxysuccinimide ester and 155 mg of ethyldiisopropylamine were added to a solution of 200 mg (0.4 mmol) of compound E.2.11 in 15 ml of dimethylformamide and the mixture was stirred at RT for 30 min , The solvent was then removed in vacuo and the residue was purified by flash chromatography on silica gel (eluent: acetonitrile / water 20/1). The appropriate fractions were combined, the solvent evaporated in vacuo and the residue dried. The Boc protective group was then removed according to standard conditions.
Yield: 75% over 2 steps [TLC: R _f = 0.22 ⁵⁾ ]. E.2.15

Production analogous to E.2.14.
Yield: 54% over 2 steps [TLC: R _f = 0.23 ⁵⁾ ]. E.2.16

Production in analogy to example E.2.14 starting from E.2.13.
Yield: 55% over 2 steps [TLC: R _f = 0.1 ⁵⁾ ]. E.2.17

Preparation by linking Boc sarcosine with compound E.2.11 in the presence of EDCI / HOBT and subsequent Boc elimination according to standard conditions.
Yield: 56% over 2 steps [TLC: R _f = 0.14 ⁵⁾ ]. E.2.18

Production in analogy to example E.2.17 starting from example E.2.12.
Yield: 71% over 2 steps [TLC: R _f = 0.1 ⁵⁾ ]. E.2.19

Preparation by linking Boc-piperidine-4-carboxylic acid with compound E.2.11 in the presence of EDCI / HOBT and subsequent Boc elimination according to standard conditions. E.2.20

Production in analogy to example E.2.19 starting from example E.2.12.
Yield: 51% over 2 steps [TLC: R _f = 0.08 ⁵⁾ ].

Examples of acid-cleavable affinity markers example 1 Manufacturing process (variant A)

45 mg (265 μmol) maleimidopropionic acid and 54 mg (0.397 mmol) 1-hydroxy-1H-benzotriazole and 61 mg (0.318 mmol) N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride were combined in 10 ml DMF and 2 h stirred at RT. 100 mg (0.265 mmol) of the compound E.2.1 and 103 mg of ethyldiisopropylamine were then added and the mixture was stirred at RT for a further 2 h. The mixture was then concentrated, the residue was taken up in dichloromethane and extracted four times with water. The organic phase was then dried over sodium sulfate and concentrated, and the residue was precipitated from dichloromethane using petroleum ether. The supernatant was decanted off and the remaining oily residue was dried under high vacuum. 103 mg (74%) of the desired product were obtained [TLC: R _f = 0.48 ⁴⁾ ].

100 mg (189 μmol) of this intermediate were taken up in 10 ml of dichloromethane and 1 ml of TFA was added. After stirring for 30 min at room temperature, the mixture was concentrated and the residue was precipitated from dichloromethane with diethyl ether. After filtration and drying, 90 mg (85%) of the desired product were obtained as the trifluoroacetic acid salt [TLC: R _f = 0.58 ⁶⁾ ].

88 mg (158 μmol) of the deprotected intermediate and 62 mg (158 μmol) of isothiocyanate E.1.1 from the educt series 1 were dissolved in 10 ml of DMF, 83 μl of ethyldiisopropylamine were added and the mixture was then stirred at RT for 3 h. It was concentrated, the residue was stirred with water and suction filtered. The residue was separated, then taken up in 4 ml of dichloromethane / methanol 1/1 and in 2 ml of DMF and precipitated with diethyl ether. The precipitate was filtered off with suction and, after drying under high vacuum, 110 mg (85%) of the target compound were obtained [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: m / e = 819 (M + H) ⁺ ]. Example 2 Manufacturing Process (Variant B)

790 mg (1.35 mmol) of compound E.2.5 were in 40 ml dichloromethane added and mixed with 8 ml of TFA. After stirring for 1 h at room temperature was concentrated and the residue was precipitated from dichloromethane with diethyl ether, which The supernatant was decanted off and the resinous product was dried under high vacuum.

800 mg (99%) of the desired product were obtained as the trifluoroacetic acid salt [TLC: R _f = 0.3 ⁵⁾ ].

790 mg (1.32 mmol) of the deprotected intermediate were placed in 30 ml of DMF and mixed with 515 mg (1.32 mmol) of the isothiocyanate E.1.1 from the educt series 1 and with 690 μl of ethyldiisopropylamine and then overnight at RT touched. It was concentrated, the residue was stirred with 10 ml of water and suction filtered. The residue was separated and then suspended in dichloromethane / methanol. 50 ml of diethyl ether were added and the precipitate was filtered off again. After filtration and drying, 1077 mg (93%) of the desired compound were obtained [TLC: R _f = 0.4 ⁴⁾ ].

1070 mg (1.22 mmol) of this intermediate were taken up in 25 ml of DMF and 1 ml of piperidine was added. After stirring for 45 min at room temperature, the mixture was concentrated and the residue was digested with dichloromethane. The mixture was then filtered off and the filter residue was suspended in a mixture of 10 ml of dichloromethane and 10 ml of methanol. After addition of 100 ml of diethyl ether, the product precipitated completely and was filtered off and dried. 805 mg (99%) of%) of the deprotected intermediate E.2.6 [TLC: R _f = 0.4 ⁶⁾ ] were obtained.

9.5 mg (45 µmol) of maleimidocaproic acid, 9.1 mg (0.067 mmol) of 1-hydroxy-1H-benzotriazole and 10.3 mg (0.054 mmol) of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride were obtained in 15 ml of DMF combined and stirred at RT for 1.5 h. Then 30 mg (0.265 mmol) of the intermediate deprotected in the precursor (compound E.2.6) and 24 μl of N, N-diisopropylethylamine were added, and the solution was then stirred at RT overnight. The solvent was evaporated and the residue was taken up in 3 ml of dichloromethane / methanol 1/1 and then treated with 15 ml of diethyl ether. The precipitated crude product was purified by flash chromatography on silica gel (eluent: acetonitrile / water 10/1). The appropriate fractions were combined, the solvent evaporated in vacuo. The residue was dissolved in dioxane / water 1/1 and lyophilized. 6 mg (16%) of the target product were obtained [TLC: R _f = 0.2 ¹⁾ ] [ESI-MS: m / e = 861 (M + H) ⁺ ].

Unless otherwise stated, the following examples were prepared in an analogous manner to example 1 (variant A) or example 2 (variant B). The respective variant is either named or described below. Example 3

Educts: E.2.2, E.1.1; option A
Yield: 11% over 3 stages
R _f = 0.65 ⁶⁾
[ESI-MS: m / e = 899 (M + H) ⁺ ]

The connection is smoothly water-soluble. Example 4

Educts: E.2.3, E.1.1; option A
Yield: 23% over 3 stages
R _f = 0.67 ⁵⁾
[ESI-MS: m / e = 861 (M + H) ⁺ ] Example 5

50 mg (75 μmol) of the compound E.2.6 were suspended in dichloromethane, and 18 μl (225 μmol) acrylic acid chloride and 12 μl pyridine were added in portions under argon. The mixture was then stirred at RT for 2 days. The solvent was evaporated and the residue was purified by flash chromatography on silica gel (eluent ¹⁾ ). The appropriate fractions were combined and the solvent was evaporated in vacuo. The residue was dissolved in dioxane / water 1/1 and lyophilized. 12 mg (22%) of the target product were obtained [TLC: R _f = 0.14 ¹⁾ ] [ESI-MS: m / e = 722 (M + H) ⁺ ]. Example 6

50 mg (75 μmol) of the compound E.2.6 were suspended in 20 ml dichloromethane, and 24 μl (300 μmol) chloroacetyl chloride and 12 μl pyridine were added under argon. The mixture was then stirred at RT for 2 h. The solvent was evaporated and the residue treated with 5 ml dichloromethane. The remaining solid was separated off and purified by flash chromatography on silica gel (eluent ¹⁾ ). The appropriate fractions were combined and the solvent was evaporated in vacuo. The residue was taken up in dichloromethane / methanol and the target product was precipitated with diethyl ether. 12 mg (22%) of the target product were obtained [TLC: R _f = 0.21 ¹⁾ ] [ESI-MS: m / e = 744 (M + H) ⁺ ]. Example 7 Manufacturing Process (Variant C)

The compound E.2.11 was reacted with Boc-glycine as described in Example E.2.5. Yield: 61% [TLC: R _f = 0.52 ⁴⁾ ].

The Boc protecting group was then removed with trifluoroacetic acid in dichloromethane. Yield: 98% [TLC: R _f = 0.2 ⁵⁾ ].

This deprotected intermediate and 62 mg (158 μmol) of isothiocyanate E.1.1 from the educt series 1 were then reacted with one another as described in Example 1 in order to obtain the target product. [TLC: R _f = 0.4 ⁵⁾ ]. Example 8

Educts: E.2.11; Boc-glycine; E.1.2 Variant C
[TLC: R _f = 0.5 ⁵⁾ ] Example 9

Educts: E.2.11; Bis-Boc-histidine; E.1.1 Variant C
[TLC: R _f = 0.35 ⁶⁾ ] Example 10

Educts: E.2.11; Bis-Boc-histidine; E.1.2 Variant C
[TLC: R _f = 0.35 ⁶⁾ ] Example 11

Educts: E.2.11; Boc-glutamic acid-δ-butyl ester; E.1.1 Variant C
[TLC: R _f = 0.5 ⁵⁾ ] Example 12

Educts: E.2.11; Boc-glutamic acid-δ-butyl ester; E.1.2 Variant C
[TLC: R _f = 0.7 ⁶⁾ ] Example 13

Educts: E.2.11; Boc-glutamic acid-δ-butyl ester; E.1.1
Variant C and subsequent transfer to the sodium salt with 1 equivalent of aqueous sodium hydroxide solution.
Yield: 38% over 4 steps [TLC: R _f = 0.4 ⁵⁾ ] [ESI-MS: m / e = 905 (M + H) ⁺ ]. Example 14

Educts: E.2.11; Boc-glutamic acid-δ-butyl ester; E.1.2
Variant C and subsequent transfer to the sodium salt with 1 equivalent of aqueous sodium hydroxide solution.
Yield: 52% over 4 steps [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: m / e = 891 (M + H) ⁺ ]. Example 15

Educts: E.2.17; Boc-glycine N-carboxylic acid; E.1.2
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 55% over 3 steps [TLC: R _f = 0.45 ⁵⁾ ] [ESI-MS: m / e = 890 (M + H) ⁺ ]. Example 16

Educts: E.2.18; Boc-glycine; E.1.2 Variant C
Yield: 37% over 3 steps [TLC: R _f = 0.4 ⁵⁾ ] [ESI-MS: m / e = 896 (M + H) ⁺ ]. Example 17

Educts: E.2.14; Boc-glycine N-carboxylic acid; E.1.2
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 47% over 3 steps [TLC: R _f = 0.45 ⁵⁾ ] [ESI-MS: m / e = 916 (M + H) ⁺ ]. Example 18

Educts: E.2.19; Boc-glycine; E.1.2 Variant C
Yield: 47% over 3 steps [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: m / e = 930 (M + H) ⁺ ]. Example 19

Educts: E.2.20; Boc-glycine; E.1.2 Variant C
Yield: 32% over 3 steps [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: m / e = 936 (M + H) ⁺ ]. Example 20

Educts: E.2.17; Boc-glycine N-carboxylic acid; E.1.1
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 55% over 3 steps [TLC: R _f = 0.46 ⁵⁾ ] [ESI-MS: mle = 904 (M + H) ⁺ ]. Example 21

Educts: E.2.17; Boc-glycine N-carboxylic acid; E.1.3
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 38% over 3 steps [TLC: R _f = 0.4 ⁵⁾ ] [ESI-MS: m / e = 961 (M + H) ⁺ ]. Example 22

Educts: E.2.17; Boc-glutamic acid-δ-butyl ester; E.1.2 Variant C
Yield: 19% over 3 steps [TLC: R _f = 0.46 ⁵⁾ ] [ESI-MS: m / e = 962 (M + H) ⁺ ]. Example 23

Educts: E.2.14; Boc-glycine N-carboxylic acid; E.1.1
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 47% over 3 steps [TLC: R _f = 0.44 ⁵⁾ ] [ESI-MS: m / e = 930 (M + H) ⁺ ]. Example 24

Educts: E.2.13; Boc-glycine; E.1.1 Variant C
Yield: 42% over 3 steps [TLC: R _f = 0.57 ⁵⁾ ] [ESI-MS: m / e = 867 (M + H) ⁺ ]. Example 25

Educts: E.2.15; Boc-glycine N-carboxylic acid; E.1.1
Variant C, with Boc-glycine-N-carboxylic acid anhydride being used as the activated amino acid component instead of activation via EDCI / HOBT.
Yield: 37% over 3 steps [TLC: R _f = 0.5 ⁵⁾ ] [ESI-MS: mle = 958 (M + H) ⁺ ].

Protein analysis studies Coupling the affinity markers to SDS-7 and description of the workflow using example 2

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

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

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

Affinity purification of derivatized peptides

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

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 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

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

30 µl sample was diluted with 30 µl 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

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

The sample was eluted with the following steps:

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

The eluate was evaporated to dryness and only shortly before analysis Mass spectrometry solved again.

PBS: stock solution 10x, 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.

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

A total of 18 different peptides from the sample were identified in an analysis, all of which carried the expected mass of the adduct with the acid-cleaved residue of the affinity marker in the same way. Not a single cysteine-containing peptide was identified that carried a complete residue of the affinity marker. Figure 2 shows an example of the peptides identified from bovine trypsin.

In Fig. 3, that the isotope-labeled affinity tag behave independently of the degree of labeling identical in their chromatographic and mass spectrometric properties is shown by the Examples 15 and 16. In Fig. 3a) the ion traces of the light and the heavy variant of the labeled peptide LFTFHADICTLPDTEKD from bovine albumin are shown as an example. There is no difference in retention time. In Fig 3b). And c) the fragment spectra of doubly charged peptide ions are shown. Both are identical except for the shift of the cysteine-containing fragments by 6 Da due to the iosotope labeling.

The same results are also obtained for other affinity markers and peptides, as shown in FIG. 4 for Examples 18 and 19. It is the APILSDSSCK peptide from bovine trypsin. Here, too, an exact coelution on the chromatography and identical behavior during fragmentation in the mass spectrometer can be observed.

Fig. 1 fragment spectrum of a derivatized with the peptide compound of Example 2 after isolation via avidin, that is acid-cleaved with affinity tag.

Fig. 2 sequence coverage of bovine trypsin, which was achieved using the compound from Example 2.

Fig. 3 MS analysis of a protein mixture after derivatization with Examples 15 and 16 in a single approach. From the ion chromatograms ( Fig. 3a)) you can see that the two variants elute at exactly the same time and occur with the same intensity. The fragment spectra of the light ( FIG. 3c)) and heavy ( FIG. 3c)) variant of the labeled peptide LFTFHADICTLPDTEK are identical except for the expected shifts of 6 Da.

Fig. 4 MS analysis of a protein mixture after derivatization with Examples 18 and 19 in a single approach. From the ion chromatograms ( Fig. 4a)) you can see that the two variants elute at exactly the same time and occur with the same intensity. The fragment spectra of the light ( Fig. 4b)) and heavy ( Fig. 4c)) variant of the labeled peptide APILSDSSCK are identical except for the expected shifts of 6 Da.

Claims (15)

1. Organic compound of the formula (I),

AL-PRG (I)

in the A for an affinity ligand residue,
PRG for a protein reactive group and
L stands for an A and PRG covalently linking linker,
characterized in that the linker L is an acid-cleavable group S of the formula


contains in the
Y for an optionally branched spacer group with a chain length of 1 to 10, preferably 1 to 5 non-hydrogen atoms and
SK stands for the side chain of an amino acid,
or their salt. 2. Connection according to claim 1, characterized in that the Spacer group Y para to the nitrogen atom on the Benzene ring is bound. 3. A compound according to claim 1 or 2, characterized in that the spacer group Y is a chain composed of the optionally substituted building blocks NH, CH ₂ and / or CO, preferably has at least NH group and is selected in particular from are NH, NH- CH ₂ and NH-CH ₂ -CH ₂ -NH-CO. 4. Connection according to one of the preceding claims 1 to 3, characterized characterized in that the protein reactive group PRG has a selective reactivity for one or more terminal functional groups of an amino acid or Phosphate group of the protein and / or for aldehyde or generated therefrom Has keto groups. 5. A compound according to any one of the preceding claims, characterized in that the protein-reactive group -PRG is selected from


6. Connection according to one of the preceding claims, characterized characterized in that the affinity ligand residue A is the residue of biotin, one Biotin derivative, a carbohydrate, a hapten, a complexing agent or a functional, a covalent fixation of the connection to one polymer matrix enabling group, in particular of biotin or one Biotin derivative. 7. Compound according to one of the preceding claims, characterized in that it is isotope-labeled for isotope coding with at least one carbon atom of the isotope ¹³ C, preferably four to 15 ¹³ C atoms. 8. A compound according to the preceding claim, characterized in that for isotope coding it is additionally isotope-labeled with at least one nitrogen atom of the ¹⁵ N isotope, preferably one to three ¹⁵ N atoms, in particular one ¹⁵ N atom. 9. A compound according to any one of the preceding claims, characterized in that it is a compound of formula (II),

ASB ¹ -X ¹ - (CH ₂ ) _n - [X ² (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -PRG (II)

in the
A the affinity ligand residue,
PRG the protein reactive group
S is the acid-cleavable group, the spacer group Y of which is preferably bonded to the benzene ring in the para position to the nitrogen atom and is particularly preferably NH, NH-CH ₂ or NH-CH ₂ -CH ₂ -NH-CO,
X ¹ , X ² , X ³ independently of one another and each X ² independently of other X ² for O, S, NH, NR, CO, CO-O, O-CO, CO-S, S-CO, SS, SO, SO ₂ , CO-NR, NR-CO, CS-NR, NR-CS, Si-O, O-Si, an arylene or diarylene group or stand for a single bond,
X ^{4 represents} O, S, NH, NR, CO-NR, NR-CO, CS-NR, NR-CS, an arylene or diarylene group, preferably NH, NR, O or S,
m, n, p, x independently of one another each represent a number from 0 to 100, the sum n + xm + p preferably being less than 100 and particularly preferably between 10 and 30,
B ^{1 stands} for an optional amine group NRR 'with the connectivity S-NRR'-X ¹ , and
R, R 'independently of one another each represent hydrogen or alkyl, alkenyl, alkynyl, alkoxy or aryl or both together with N represent an N-heterocycle NRR', where R 'is additionally bonded to X ¹ and therefore cannot stand for hydrogen. 10. Connection according to one of the preceding claims, characterized characterized in that they have a protein reactive group PRG according to claim 5 and a linker L according to claim 9, wherein the sum of n + xm + p and q, z, r or s is preferably less than 100 and especially is preferably between 5 and 30. 11. A process for the preparation of a compound of formula (1I) according to claim 9 or 10, in which a) a compound of the formula (III),

HB ¹ -X ¹ - (CH ₂ ) _n - [X ² (CH ₂ ) _m ] xX ³ - (CH ₂ ) _p -X ⁴ -V (III)

in which V represents a hydrogen atom or a hydroxyl group and the other variables have the same meaning as in formula (II), first reacted with an amino acid derivative protected on the N-terminal amino group to give a conjugate of formula (IV) .

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - (X ² [CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (IV)

where SG stands for a protective group and SK for the side chain of an amino acid, b) then the conjugate of the formula (IV) with the derivative of a protein-reactive group or the activated precursor of a protein-reactive group of the formula (V),

U-PRG (V)

in which U stands for a group which enables the linkage of PRG with X4, for example by being converted into a leaving group together with the radical V of the conjugate of the formula (IV), c) the amino protective group SG is then removed, a conjugate of the formula (VI)

H ₂ NCH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p X ⁴ -PRG (VI)

will get d) the affinity ligand A-OH or an activated form thereof with a compound of the formula (VII),


in which Y stands for the optionally branched spacer group, which can optionally carry a protective group, for the compound of the formula (VIII)


is implemented e) the compound of the formula (VIII) is then converted into a corresponding isothiocyanate after prior elimination of an optionally introduced protective group, f) the isothiocyanate is then coupled with the conjugate of the formula (VI) to the thiourea of the formula (IX) and


g) any protective groups still present are split off in an optional last step, the successive steps iv) and v) can be carried out at any time before step vi). 12. A process for the preparation of a compound of formula (II) according to claim 10, in which a) a compound of the formula (III),

HB ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (III)

in which V represents a hydrogen atom or a hydroxyl group and the other variables have the same meaning as in formula (II),
first reacted with an amino acid derivative protected on the N-terminal amino group to give a conjugate of the formula (IV),

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -V (IV )

where SG stands for a protective group and SK for the side chain of an amino acid, b) the conjugate of the formula (IV) by introducing a further protective group SG 'into the derivative of the formula (XIII)

SG-NH-CH (SK) -CO-B ¹ -X ¹ - (CH ₂ ) _n - [X ² - (CH ₂ ) _m ] _x -X ³ - (CH ₂ ) _p -X ⁴ -SG '( XIII)

transferred and then the N-terminal protecting group SG is released, c) the affinity ligand A-OH or an activated form thereof with a compound of the formula (VII),


in which Y stands for the optionally branched spacer group, which can optionally carry a protective group, for the compound of the formula (VIII)


is implemented d) the compound of the formula (VIII) is then converted into a corresponding isothiocyanate after prior elimination of an optionally introduced protective group, e) the isothiocyanate with the derivative of the formula (XIII) to the conjugate of the formula (XIV)

ASB ¹ -X ¹ - (CH ₂ ) _n [X ₂ - (CH ₂ ) _m ] _x -X ² - (CH ₂ ) _m -X ³ - (CH ₂ ) _p -X ⁴ -SG '(XIV)

is implemented f) the conjugate of the formula (XIV) after detachment of the protective group SG 'with the derivative of a protein-reactive group or the activated precursor of a protein-reactive group of the formula (V),

U-PRG (V)

in which U represents a group which enables PRG to be linked to X ⁴ , for example by becoming a leaving group together with the radical V in the conjugate of the formula (IV),
is coupled to the thiourea of the formula (IX) and


g) any protective groups still present are split off in an optional last step, the successive steps iii) and iv) can be carried out at any time before step v). 13. Use of one or more differently labeled isotopes Compounds according to one of the preceding claims as a reagent for mass spectrometric analysis of proteins. 14. Use according to the preceding claim for the identification of one or more proteins or protein functions in one or several 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 samples.