DE10060746A1 - New divalent or trivalent linking agents, useful for dimerizing proteins and immobilizing them on carriers, comprise thiol-binding group and sulfur-containing functional group - Google Patents

Abstract

Di- and tri-valent linkers (I) comprise at least one thiol-binding group and a thio or disulfide functional group. Di- and tri-valent linkers of formula (I) comprise at least one thiol-binding group and a thio or disulfide functional group: Y = CR<1> or N; R<1> = H or 1-5C alkyl; Q = sulfur-containing coupling group; X = group reactive with thiol; S' = H, 1-5C alkyl or -Z<3>X; Z<1>- Z<3> = (CH2)n, in which one or more non-adjacent methylenes can be replaced by NR<1>, O, CO, CH=CH, acetylenylene, NR<1>CO, CONR<1>, OCO, COO, NR<1>COO, OCONR<1>, NR<1>CONR<1>, CHR<2>, NR<1>CHR<2>, CHR<2>NR<1>, CONR<1>CHR<2>, CHR<2>NR<1>CO, COOCHR<2> or CHR<2>OCO; and R<2> = OH, amino, carboxy or 1-5C alkyl. Independent claims are also included for: (a) method for preparing (I); (b) use of (I) for covalent dimerization of proteins to form conjugates (C); and (c) immobilization of (C) on solid surfaces.

Description

The invention relates to the synthesis of bi- or tridental linkers that at least one thiol-binding group and one thiol or disulfide Functionality included, as well as the use of these linkers Preparation of conjugates.

The coupling of biological substances to macromolecules or to the base Carrier is made using bifunctional linkers or, if additional links should be achieved with trifunctional Left. Thus EP-A-0 446 071 discloses trifunctional linkers with three Maleimide groups. Such linkers are for binding and networking of biological substances not very suitable, because uncontrolled Networks are expected. EP-A-0 618 192 discloses trifunctional Linker with two maleimide groups and an activated ester group. Maleimide groups have specificity for thiol groups, as described in native proteins or by mutation or derivatization can be introduced. However, the activated ester function of the in EP-A-0 618 192 disclosed linkers with a very wide reactivity. she binds, among other things, to amino or hydroxyl groups, such as basic can also be found in mostly large numbers of biological substances are. In particular, such groups can be found in the active centers of enzymes. Such linkers react with groups from the active center, so the enzymatic activity in general blocked. So the problem of uncontrolled networking is only partially avoided.

The object of the present invention was therefore to be difunctional or trifunctional To provide linkers, on the one hand, through the reaction of their thiol-binding functions a defined, reproducible coupling of  Proteins enable, on the other hand, an additional selective one Allow connection to carrier materials.

To ensure selective binding to carrier materials, in the linkers according to the invention contain sulfur-containing groups, such as for example SH groups. These functionalities are however, as can be seen immediately, not entirely with the Use of maleimide groups or other thiol-reactive Groups tolerated. By providing the linker according to the invention, and the method for their production, this problem is solved.

The task is performed by compounds of the general formula I.

wherein
Y is a core component selected from the group CR ¹ or N, where
R ¹ = H or branched or unbranched alkyl having 1-5 C atoms,
Q is a sulfur-containing coupling group,
X is a group reacting with thiols,
S 'H or a branched or unbranched alkyl having 1-5 C atoms or Z ³ -X
Z ¹ , Z ² , Z ³ independently of one another bridge members of the form (CH ₂ ) _m with m = 1-50, in which one or more non-adjacent methylene groups by -NR ¹ -, -O-, -CO-, -HC = CH -, -C = C-, -NR ¹ CO-, -CONR ¹ -, -O-CO-, -CO-O-, -NR ¹ CO-O-, -O-CONR ¹ -, -NR ¹ CONR ¹ -, -CHR ² -, -NR ¹ -CHR ² -, -CHR ² -NR ¹ -, -CO-NR ¹ -CHR ² -, -CHR ² -NR ¹ -CO-, -CO-O-CHR ² -, -CHR ² -O- CO-, with
R ² = OH, NH ₂ , COOH, branched or unbranched alkyl with 1-5 C atoms, can be replaced.

The invention therefore relates to compounds of the general Formula I, which in addition to one or two groups reacting with thiols carry another sulfur-containing coupling group.

Compounds of the general formula I in which S '= Z ³ -X are particularly preferred.

Furthermore, particular preference is given to compounds of the general formula I in which S '= Z ³ -X and the core component Y is either CR ¹ with R ¹ = H or Y = N.

The invention also relates to the preparation of compounds of the general formula I, where S '= Z ³ -X and the core unit Y = CR ¹ with R ¹ = H, by

• a) double malonic ester synthesis of compounds of formula II Malonic acid diesters to compounds of formula III
• b) Cleavage of the phthalimido groups and release of the amino functions
• c) Cleavage of the malonic acid diester and decarboxylation
• d) Introduction of amino protecting groups
• e) conjugation of the carboxylic acid with amino compounds of the form H ₂ NZ ² -Q.
• f) cleavage of the amino protective groups
• g) Introduction of two maleimidyl residues by treatment with Alkoxycarbonylmaleimide.

The invention furthermore relates to the synthesis of compounds of the general formula I, where S '= Z ³ -X and the core unit Y = N, by

• a) Introduction of amino protective groups into a molecule of the form H ₂ NZ ¹ - NH-Z ³ -NH ₂
• b) Conjugation of a carboxylic acid linker of the form HOOC-Z ² -Q to the central secondary amine
• c) cleavage of the amino protective groups
• d) Introduction of two maleimidyl residues by treatment with Alkoxycarbonylmaleimide.

The invention also relates to the use of a Compound according to the invention for the covalent dimerization of Proteins, whereby the conjugation takes place via the groups X of the linker.

The invention also relates to the immobilization of such Conjugates via the sulfur-containing coupling group on solid phases surfaces.

The invention also relates to the construction of dendritic structures Carrier materials by means of linker modules according to the invention and the Immobilization of proteins on the surface structures.

In Fig. 1 and 2, a general synthetic route for compounds according to claim 3 is shown, where the bridge members Z ¹ and Z ³ contain only methyl groups.

In Fig. 3 and 4, a general synthetic route for compounds according to claim 4 is shown, the bridge members Z ¹ and Z ³ contain only methyl Grupen.

Fig. 5 shows a general scheme for the immobilization of proteins on support surfaces.

Fig. 6 shows the example of a surface covered with dendritic structures.

It has been found that covalent linkages of proteins and the later defined immobilization on the solid phase, as determined by the Linker invention are made possible, a great advantage compared to the state of the art. A variety of Enzymes consist of several, e.g. two, protein sub units that only have hydrophobic and / or ionic interactions be held together. Such enzymes are often in one sensitive dissociation-reassociation equilibrium and already dissociate due to small changes in pH, Changes in ionic strength or the presence of detergents or chaotropic salts in the surrounding medium. Most are the monomers Subunits of the oligomeric enzymes are catalytically inactive.

In conventional methods for immobilizing oligomeric proteins the enzymes are often only linked to a carrier matrix via the covalent connection of a subunit. Therefore one observes as a result the dissociation of the subunits often an irreversible reduction in the enzymatic activity or a washout of enzymes.

The linkers according to the invention enable the covalent linking of dimeric enzymes and their subsequent immobilization without reduction the enzymatic activity. Immobilization takes place according to the invention  via a sulfur-containing coupling group, which has a selective connection allowed to appropriately functionalized carriers. That way it is possible, for example for biosensors, chromatography materials or Enzyme membranes, always new, for the respective application to develop specially adapted linkers that allow two Protein subunits or two structurally different proteins connect covalently and defined to a given matrix immobilize. This method allows the implementation in more detail Studies on cooperative effects of proteins, dimerized proteins or protein subunits for their enzymatic activity. Is a previous dimerization is not important in the application, can the linker according to the invention for immobilization in a heterobifunctional, bidental form can be selected.

The heterotrifunctional linkers of the present invention can also used to build dendritic surface structures on matrices become. Convergent and divergent syntheses can be carried out. The The sulfur-containing group of linkers is not only used for direct purposes Connection to the matrix. At the same time there is the possibility to react with the thiol binding groups of other linker molecules. That’s what happens to form a dendritic space-filling surface covering. This greatly enlarged surface can in turn be used to immobilize Proteins or dimerized proteins or also for loading with Peptides or low molecular weight affinity markers can be used. The dendritic structures allow very high levels of loading to reach.

The representation of dimers is equally feasible Protein conjugates using the linker according to the invention, a subsequent release of the SH function and implementation with others Linker molecules. The dendritic structures created in this way can can also be used to assign appropriate matrices.  

The core building block from which the arms of the linker originate is nitrogen or carbon used. The carbon can be alkyl substituted or preferably carry a hydrogen atom. The core building block as linker arms outgoing bridge links can be in length and Polarity of the respective task can be adjusted. Your Chain length can range from 1 to 50 methylene groups. Next the preferred methylene groups can also be used once or multiple functionalities, such as ether, carbonyl, acetyl, amide, ethylene, Acetylene, amine, urethane, urea or substituted methylene groups be inserted.

Substituted are preferably substituted as sulfur-containing coupling groups Disulfide used. The radicals S-3-nitro- are particularly preferred. 2-pyridine-sulfenyl-, S-alkoxycarbonyl-sulfenyl- or S-tert-butylmercapto-. The use as ether or ester-protected thiols is also possible, as the expert from T. Green, Protective Groups in Organic Synthesis, J. Wiley & sons inc., Second ed. 1991, ISBD 0-471-62301-6, 279-307 are known.

As the group reacting with thiols, preference is given to the maleimidyl and the 2-pyridyl group or also haloacetyl radicals, of which Iodoacetyl is particularly noteworthy.

Preferred targets for protein dimerization are Mercapto groups from cysteine side chains. You can also do this by targeted mutation cysteine residues are introduced into a protein or Mercapto groups can be generated with reagents such as 2-iminothiolane.

The maleimide groups on the trifunctional linkers according to the invention react highly selectively with SH groups to form a stable Thioether bond. Carrying out the reaction is particularly advantageous  at pH values between 6.5 and 7.5. Is done above pH 7.5 increasingly also a reaction with primary amines. That way bound proteins can be proteins of the same or different types It can be protein subunits, multienzyme complexes or Act peptide fragments.

The covalently bound proteins are immobilized via the sulfur-containing coupling group. A disulfide function also allows that direct binding of the linker to gold surfaces. In a reduced condition the SH group is used for selective binding to functionalized Surfaces such as membranes, magnetic particles, hollow fibers, electrodes, Glass, chromatography gel or polymers. The functionalization takes place preferably with maleimide groups, 2-pyridyl, haloacetyl or haloacyl Leftovers.

The synthesis of linkers according to the invention in which S '= Z ³ -X and Y = CH, where the said radicals S', X, Y and Z ^{3 have} the meaning given in formula I, can be carried out by processes which are known in principle, for example by double malonic ester synthesis with molecules of the formula II, where G is a halogen, preferably bromine, and malonic diesters, preferably malonic diethyl ester.

G = Cl, Br, J
Z ¹ has the meaning given in claim 1.

R = branched or unbranched alkyl with 1-5 C atoms

Molecules corresponding to formula III are obtained. After the amino groups have been released by splitting off the phthalimido groups (preferably using hydrazine), the malonic acid diester is split off (preferably using hydrochloric acid) and decarboxylation is carried out. The primary amino groups are then protected, for which purpose a t-Boc group is preferred. An amino compound of the form H ₂ NZ ² -Q is then conjugated to the carboxylic acid function. After the protected amino groups have been released again, two maleimidyl residues are introduced by treatment with alkoxycarbonylmaleimide, preferably N-methoxycarbonylmaleinimide.

The introduction of other groups that react with thiols instead of Maleimides can be carried out according to known methods. The basics of such synthetic methods and more suitable Process variants are known to the person skilled in the art. The used Starting substances are commercially available or in the literature described.

The synthesis of linkers according to the invention with S '= Z ³ -X and Y = CH, where the said radicals S', X, Y and Z ^{3 have} the meaning given in formula I, can be carried out starting from a molecule of the form H ₂ NZ ¹ -NH-Z ³ -NH ₂ . After protecting the primary amino groups (preferably with t-Boc), the central secondary amino group is conjugated with a carboxylic acid linker of the form H ₂ NZ ² - Q. After the amino protecting groups have been split off, the maleimide group can then be introduced by treatment with alkoxycarbonylmaleinimide, preferably N-methoxycarbonylmaleinimide become.

Fig. 5 shows a general scheme for the dimerization and immobilization of a protein, such as the S140C-Sma nuclease, using a crosslinker according to the invention. The linker 5-maleimido-2- (3- (maleimido) -propyl) -pentanoic acid- (2-tert-butyldisulfanyl-ethyl) -amide was chosen as an example. In step A, the enzyme subunits are first dimerized. In step B, the SH group is released. Finally, in step C, the immobilization is carried out on a maleimide-functionalized carrier material.

Fig. 6 shows schematically tree-like, dendritic spacer structures that were produced with the help of the trifunctional linker according to the invention. The strength of the crosslinking can be adjusted by appropriate reaction conditions.

Even without further explanations, it is assumed that a subject man can use the above description to the widest extent. The preferred embodiments and examples are therefore only as descriptive, in no way as in any way limiting to understand the agreement.

The complete disclosure of all those listed above and below Applications, patents and publications, especially the corresponding application DE 199 61 701, filed on December 21, 1999, is incorporated by reference into this application.  

In the examples below, the abbreviations mean:
EDAC = 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
HOBT = 1-hydroxybenzotriazole

example 1 1.1 Diethyl bis (3-phthalimidopropyl) malonate literature

A. Reissert, Chem. Ber. 26 (1893) 2137.

9 g (0.39 mol) of sodium were added portionwise and carefully with stirring dissolved in 108 ml of dried ethanol. To the resulting milky white solution were 28.5 ml (0.19 mol) at RT with vigorous stirring Diethyl malonate in 30 ml of dried ethanol was added dropwise. The The reaction solution was diluted twice with 50 ml of ethanol and stirred until a clear solution was found.

100 g (0.37 mol) of solid, undissolved N- (3- Bromopropyl) phthalimide was added in portions with vigorous stirring and the reaction solution was refluxed overnight.

The white product which precipitated from the reaction solution was washed twice with 100 ml of water and then twice with 100 ml of hot ethanol. The reaction product was dried over CaCl ₂ in a vacuum desiccator.

54.7 g of white crystals (55% of theoretical yield) are obtained with a Melting point of 156.9 ° C.

1.2 2- (3-aminopropyl) -5-aminopentanoic acid literature

S. Löfås and P. Ahlberg, J. Heterocyclic Chem. 21 (1984) 583.

A solution of 53.46 g (0.1 mol) of bis (3-phthalimidopropyl) - diethyl malonate in 500 ml ethanol was treated with 9.7 ml (0.2 mol 100 % hydrazine hydrate added and heated under reflux for 1.5 h. The Solvent was evaporated after adding 100 ml of water and the Residue in 300 ml of concentrated hydrochloric acid for 12 h under reflux heated.

The reaction mixture was diluted twice, filtered and in vacuo to constricted to the dry. The product was dissolved in 200 ml of 1 M sodium hydroxide solution neutralized. The solvent was removed and the product without further workup used in the subsequent reaction.

1.3 2- (3-BOC-aminopropyl) -5-BOC-aminopentanoic acid

17.4 g (0.1 mol) of 2- (3-aminopropyl) -5-aminopentanoic acid were dissolved in 250 ml of 1 M NaOH and 250 ml of tert-butanol were added. 43.6 g (0.2 mol) of di-tert-butyl dicarbonate were added dropwise to this emulsion at RT with stirring. The white solid which precipitated out of the reaction mixture was filtered off and the remaining emulsion was extracted three times with 200 ml of n-pentane. The aqueous phase was adjusted to pH 3 with a 1.1 M solution of KHSO ₄ and extracted three times with 200 ml of diethyl ether, the ethereal phase was washed with water, dried over sodium sulfate and evaporated. 25.4 g of a white powder (68% of theoretical yield) are obtained.

1.4 2-Aminoethyl-2-aminoethanethiol sulfonate dihydrochloride literature

Field, L. et al., J. Am. Chem. Soc., 83 (1961) 4414-4417.

To a well-stirred 40 g (0.35 mol) solution cooled to 0 ° C Cysteaminium chloride in 100 ml water was 50 ml (0.58 mol) Hydrogen peroxide (35%) added dropwise. (some were used as a catalyst  few crystals of potassium iodide added). The reaction mixture was stirred for 1 h at 0 ° C and then for a further 20 h at RT. The solvent was removed in vacuo and the yellowish Recrystallized from 250 ml of glacial acetic acid.

The white precipitate was filtered off and dried in vacuo. 42.4 g of a white crystalline substance (94% of theor. Yield), which decomposes at 162 ° C and is very soluble in water.

1.5 2- (tert-Butyldithio) ethylamine hydrochloride literature

Field, L. et al., J. Am. Chem. Soc., 83 (1961) 4414-4417.

To a well-stirred solution of 51.4 g (0.2 mol) at a temperature of 25 ° C 2-aminoethyl-2-aminoethanethiol sulfonate dihydrochloride in 100 ml of water 18 g (0.2 mol) of tert-butyl mercaptan in 50 ml of ethanol became slow dripped. The reaction mixture was stirred at 25 ° C. for a further 1 h and then left overnight at 4 ° C.

The white, crystalline precipitate was filtered off and the filtrate was evaporated at 40 ° C. in vacuo. The white residue was taken up in 100 ml of water, mixed with 100 ml of diethyl ether and neutralized with KHCO ₃ .

The ether phase was extracted with 8.5 ml of 37% HCl in 50 ml of water and washed with 100 ml of water. The acidic extract and the Wash solution were combined and concentrated in vacuo. The white solid The residue was recrystallized from isopropanol / hexane.

14.4 g of the product are obtained (36% of theoretical yield).

1.6 2- (tert-Butyldithio) ethylamine

14.25 g (0.07 mol) of 2- (tert-butyldithio) ethylamine hydrochloride were added to 100 ml of 1 M aqueous sodium bicarbonate solution was added and 5 min  stirred at RT. The cloudy solution was then 3 × 100 ml Extracted diethyl ether. The combined ether phases were over Dried sodium sulfate and then the solvent in vacuo deducted.

11.4 g of a colorless liquid are obtained as product (98% of theor. Yield).

1.7 N- (2- (tert-Butyldithio) ethyl) -2- (3-BOC-aminopropyl) -5-BOC- aminopentanoic acid amide

10.2 g (27.2 mmol) of 2- (3-BOC-aminopropyl) -5-BOC-aminopentanoic acid and 8.4 g (43.6 mmol) EDAC were added in 150 ml dichloromethane with stirring RT solved. After a clear solution had formed, 7.4 g (54.5 mmol) HOBT were added and the mixture was stirred at RT for a further 30 min.

4.5 g (27.2 mmol) of 2- (tert.- Butyldithio) ethylamine added and stirred overnight at RT.

The reaction mixture was treated with 3 × 100 ml of 0.5 M HCl, then with 3 × 0.5 M sodium carbonate solution and finally with 2 × 100 ml of water washed. The remaining yellow colored organic phase was over Dried sodium sulfate, the solvent removed in vacuo and the oily residue taken up in 30 ml of ethyl acetate. The solution was Chromatographed on a glass filter funnel filled with silica gel 60. The eluate was left overnight at -20 ° C. The White The precipitate was washed several times with n-hexane. The result is 8.4 g of a white powder (59% of theoretical yield).

1.8 N- (2- (tert-butyldithio) ethyl) -2- (3-aminopropyl) -5- aminopentanoic acid amide

2 g (3.8 mmol) N- (2- (tert-butyldithio) ethyl) -2- (3-BOC-aminopropyl) -5- BOC-aminopentanoic acid amide were stirred at RT with stirring in 20 ml  anhydrous trifluoroacetic acid (TFA) dissolved and stirring continued for 2 h at RT. The TFA was then evaporated in vacuo at 40 ° C. The The residue was taken up twice with 40 ml of toluene and in Vacuum removed.

The residue was dissolved in 100 ml of 0.5 M sodium carbonate solution recorded and extracted with 3 × 100 ml dichloromethane. The United organic phases were dried over sodium sulfate and the Solvent evaporated in vacuo. The oily residue (0.65 g) was used directly in the subsequent reaction.

1.9 5-Maleimido-2- (3- (maleimido) propyl) pentanoic acid- (2-tert.- butyldisulfanyl-ethyl) -amide

10 mmol of N- (2- (tert-butyldithio) ethyl) -2- (3-aminopropyl) -5- aminopentanoic acid amide in 90 ml of saturated NaHCO ₃ solution / THF (1: 1 v / v, approx. 9 ml of solvent per millimole of diamino compound). The solution was cooled to 0 ° C. and 3.7 g (24 mmol; 2.4 equiv.) Of N-methoxycarbonylmaleimide were added in portions with stirring.

The reaction mixture was stirred at 0 ° C. for 10 min and a further 90 ml of the NaHCO ₃ / THF mixture were added. The reaction mixture was then stirred for a further 3 h at RT, 90 ml of the NaHCO ₃ / THF mixture being added every hour in order to keep the solution basic. The reaction solution was extracted with 3 × 100 ml of ethyl acetate. The combined organic phases were washed 2 × with 100 ml of water and 1 × 100 ml of saturated NaCl solution and dried over sodium sulfate. The solvent was evaporated in vacuo at 40 ° C. The viscous product was taken up in 20 ml of n-hexane and crystallized with a few milliliters of methylene chloride. The yield after recrystallization in ethanol was 1.0 g (21% of theoretical yield); Melting point: 113.6 ° C.

Claims (10)

1. Compounds of the general formula
wherein
Y is a core component selected from the group CR ¹ or N,
R ^{1 is} H or branched or unbranched alkyl having 1-5 C atoms,
Q is a sulfur-containing coupling group,
X is a group reacting with thiols,
S 'H or a branched or unbranched alkyl having 1-5 C atoms or Z ³ -X
Z ¹ , Z ² , Z ³ independently of one another bridge members of the form (CH ₂ ) _m with m = 1-50, in which one or more non-adjacent methylene groups by -NR ¹ -, -O-, -CO-, -HC = CH -, -C = C-, -NR ¹ CO-, -CONR ¹ -, -O-CO-, -CO-O-, -NR ¹ CO-O-, -O-CONR ¹ -, -NR ¹ CONR ¹ -, -CHR ² -, -NR ¹ -CHR ² -, -CHR ² -NR ¹ -, -CO-NR ¹ -CHR ² -, -CHR ² -NR ¹ -CO-, -CO-O-CHR ² -, -CHR ² -O- CO-, with
R ² = OH, NH ₂ , COOH, branched or unbranched alkyl having 1-5 C atoms,
can be replaced
mean. 2. Compounds according to claim 1, in which S '= Z ³ -X, where the radicals X and Z ^{3 have} the meaning given in claims 1. 3. Compounds according to claim 2, in which the core block Y = CR ¹ with R ¹ = H. 4. Compounds according to claim 2, in which the core component Y = N means. 5. A process for the preparation of compounds according to claim 3

• a) double malonic ester synthesis of compounds of the formula II, where G = Cl, Br, J, Z ¹ has the meaning given in claim 1,
  on malonic diesters to give compounds of the formula III, where R is a branched or unbranched alkyl having 1-5 C atoms
  
• b) Cleavage of the phthalimido groups and release of the amino functions
• c) Cleavage of the malonic acid diester and decarboxylation
• d) Introduction of amino protecting groups
• e) conjugation of the carboxylic acid with amino compounds of the form H ₂ NZ ² -Q, where the radicals Z ² and Q have the meaning given in claim 1
• f) cleavage of the amino protective groups
• g) Introduction of two maleimidyl residues by treatment with alkoxycarbonylmaleimide.

6. A process for the preparation of compounds according to claim 4

• a) Introduction of amino protective groups into a molecule of the form H ₂ NZ ¹ - NH-Z ³ -NH ₂ , where the radicals Z ¹ and Z ^{3 have} the meaning given in claim 1.
• b) conjugation of a carboxylic acid linker of the form HOOC-Z ² -Q, where the radicals Z ² and Q have the meaning given in claim 1, to the central secondary amine
• c) cleavage of the amino protective groups
• d) Introduction of two maleimidyl residues by treatment with alkoxycarbonylmaleimide.

7. Use of a compound according to claim 2 for covalent Dimerization of proteins, the conjugation via groups X of the linker. X has the meaning given in claim 1. 8. Immobilization of the conjugates obtained according to claim 7 Solid phase surfaces. 9. Use of the compounds according to claim 2 for construction dendritic structures on carrier surfaces. 10. Use of the surface structures generated according to claim 9 for Immobilization of proteins.