DE3645104C2 - New maleimido derivs. of biotin hydrazide - Google Patents

Abstract

Biotin derivs. of formula (I) are new.R1 = a spacer, pref. an aliphatic, aromatic or aromatic-aliphatic gp., opt. selectively cleavable by chemical or enzymatic methods.Specifically, R1 = (CH2)n (n = 1-5, esp. 2-3); phenylene; p-phenylene(-CH2-)n (the phenyl being bonded to maleimido) or CHR2 (R2 = amino acid side chain, esp. Ala, Met-sulphone or Cys)

Description

The invention relates to a method for producing Biotin derivatives of thiols using a biotin derivative where a maleimido group on biotin hydrazide is bound.

The "biotin-avidin system" is widely used in molecular biology and immunology. Many are known Methods for coupling biotin to amino, Carboxy, phenol, imidazole or thiol groups or Sugar residues (Methods of Biochemical Analysis (1980) 26, 1-45). So far, biotinylation of SH groups has been used only the relatively poorly soluble bromoacetyl or p-hydroxy mercuribenzoyl Derivatives of biotin are used.

However, biotin has also been Derivative of biotinyl-lysine, 3- (N-maleimidopropionyl) - Biocytins, coupled to thiol groups (Analytical Biochemistry (1985), 529-536).

The coupling of the biotin to SH groups of proteins is especially advantageous when coupling to lysine residues the biological to form an amide bond Protein activity impaired.

The invention has for its object an improved To provide methods for biotinylation.

This task was solved by using maleimido Group-bearing derivatives of biotin hydrazide - abbreviated to MBH derivatives.  

Surprisingly, it was found that the comparison with the prior art (3- (N-maleimido-propionyl) biocytin; Analytical Biochemistry (1985) 149, 529-536) simpler biotin hydrazide derivatives of the formula I are suitable for the production of biotin derivatives which by reaction with avidin for biochemical analysis can be used. These connections react with SH groups.

The invention relates to a method for manufacturing a biotin derivative of a thiol, one Compound of formula I.

wherein
R¹ - (CH₂) _m - with m = 1 to 5 or
a spacer of formula III

-R²- (CH₂) _n - (III)

is what
n 1 to 5
R² is p-phenylene or
R¹ is a radical of the formula IV,

-CHR³- (IV)

wherein
R³ means the side chain of alanine, serine, threonine, methionine, methionine sulfone, cysteic acid, aspartic acid or glutamic acid, in dimethylformamide or dimethylsulfoxide or in a mixture of dimethylformamide or dimethylsulfoxide with water or a buffer, with a solution of the thiol and together with a pH of 5 to 9 can react and the biotin derivative thus prepared is purified and isolated.

For the purposes of the invention, thiols are preferably molecules, such as B. proteins or peptides, which are naturally present, wear introduced or generated SH groups. However, thiols are also matrices carrying SH groups.

Is used in the implementation of the biotin hydrazide with a Maleimido compound of formula II the reaction so leads to the maleimido compound completely consuming then the compound of formula I containing reaction mixture without further purification Biotinylation of SH groups can be used.

Excess biotin hydrazide and excess compounds of the formula I can be used after the implementation SH groups of macromolecules such as proteins or particles such as cells, for example by dialysis, ultrafiltration or remove washes.

The compound of formula I is preferred for biotinylation of SH groups, however, as a purified compound used.

Compounds of formula I can be used in a variety of ways will. They are universal tools for indirect Marking of SH groups using marked Avidin. For example, there can be a Fluorescence, luminance, enzyme or radioactivity labeling be used.  

For example, it can be completely immunoreactive Prepare antibody conjugates by inserting into the Fab 'fragment or biotinylated the "hinge" SH groups in the antibody. Such derivatives can be used in immunological determination reactions be used. Labeled avidin can before, after or during the course of the immune reaction be added. Has a biotinyl Fab 'molecule because of its small size compared to one Whole-Antibodies the advantages of a fast reaction process, of faster penetration into cavities and faster penetration of narrow channels as well as the absence of unwanted reactions via the Fc part. This Methods are also suitable for linking different ones Antibody specificities (chemical antibodies).

For example, a marking component like that Enzyme β-galactosidase by converting its SH groups can be directly biotinylated. A marker enzyme like that Peroxidase can, for example, after the introduction of SH groups, for example by reaction with 2-iminothiolane, be biotinylated. Such biotinylated enzymes can in an immunoassay using the avidin-biotin Systems as described in Immunology Today 5, 39-45 (1984) described, used.

Compounds of the formula I are also suitable for biotinylation from existing ones, for example from a disulfide generated or introduced SH groups on the Surface of molecules, cells, organelles, particles or receptors so that these can be labeled via avidines can be proven and determined.

For example, rabbit anti-HCG antibodies after reduction of the S-S double bonds of the "hinge" region with a compound of formula I, wherein R¹ (CH₂) ₂, (CH₂) ₃ or CH (CH₃) is biotinylated.  

The following examples are intended to illustrate the invention.

Example 1 Preparation of β-maleimido-propionyl-biotin hydrazide (β-MPBH) (compound of the formula I with R¹ = -CH₂-CH₂-)

8.4 mg (0.033 mmol) biotin hydrazide and 10.5 mg (0.038 mmol) Compound of formula II with R¹ = -CH₂-CH₂- and Succinimidyloxy as the activating group was in 1 ml dry dimethylformamide dissolved under heating and Stirred overnight at room temperature. For saponification Excess compound of formula II were 0.1 ml Water was added and the solution after a further 3 h Rotary evaporator evaporated to dryness in an oil pump vacuum. The residue was made up in 1 ml of a mixture Suspended methylene chloride / methanol (8/2), the insoluble Filtered β-MPBH and dried in vacuo. additional β-MPBH was removed from the filtrate by dropwise addition precipitated from petroleum ether and filtered off. The received β-MPBH (about 11 mg) was used without further purification Biotinylation of thiol groups used.

To make β-MPBH pure, the crude product was dissolved in 0.5-1 ml of dimethylformamide by heating, diluted with 0.5-1 ml of methylene chloride / methanol (4/1) and poured over a silica gel 60 (0.063-0.200 mm, Merck) - Column (filling height 16 cm, diameter 1 cm) at maximum possible dropping speed with methylene chloride / methanol (4/1) as eluent separated and identified by thin layer chromatography in an iodine chamber. Pure β-MPBH in a mixture of D₆-dimethyl sulfoxide and deuterochloroform against tetramethylsilane as the internal standard showed the following 400 MHz 1 H-nmr spectrum: 1.2-1.8 (m, 6H; CH₂-9, CH₂-10, CH₂- 11), 2.16 (t, 2H, J = 7.2 Hz, CH₂-12), 2.46 (t, 2H, J = 7.4 Hz, CH₂-17), 2.64 (d, 1H , J _{2.2 ′} = 12.4 Hz, H-2), 2.83 (dd, 1H, J _{2.2 ′} = 12.4 Hz, J _{2 ′, 3} = 5.0 Hz, H-2 ′), 3.10 (m, 1H, H-8), 3.68 (t, 1H, J = 7.5 Hz, CH₂-18), 4.18 (m, 1H) and 4.35 (m , 1H) H-3 and H-7, 6.33 (s, 1H) and 6.37 (s, 1H) H-4 and H-6, 6.90 (s, 2H, maleimido-H), 9 , 74 (s, 1H, H-14), 9.89 (s, 1H, H-15).

Example 2 Preparation of gamma-maleimido-butyryl-biotin hydrazide (gamma-MBBH) (compound of formula I with R¹ = -CH₂-CH₂-CH₂-)

5.6 mg (0.022 mmol) biotin hydrazide and 7.4 mg (0.026 mmol) Compound of formula II with R¹ = -CH₂CH₂CH₂- and Succinimidyloxy as the activating group was in 1 ml dry dimethylformamide dissolved under heating and worked analogously to Example 1. The gamma-MBBH obtained (5.7 mg) became biotinylation without further purification used by thiol groups.

The crude product was chromatographed analogously to Example 1 for the purification of gamma-MBBH. 1H-nmr (400 MHz) in D₆-DMSO / CDCl₃: 1.2-1.8 (m, 6H; CH₂-9, CH₂-10, CH₂-11), 1.80 (m, 2H, CH₂-18 ), 2.16 (t, 4H, CH₂-12 and CH₂-17), 2.65 (d, 1H, J _{2.2 '} = 12.5 Hz, H-2), 2.84 (dd, 1H , J _{2.2 ′} = 12.5 Hz, J _{2 ′, 3} = 5.0 Hz, H-2 ′), 3.10 (m, 1H, H-8), 3.48 (t, 1H, J = 7.5 Hz, CH₂-19), 4.18 (m, 1H) and 4.36 (m, 1H) H-3 and H-7, 6.34 (s, 1H) and 6.39 ( s, 1H) H-4 and H-6, 6.88 (s, 2H, maleimido-H), 9.74 (s, 2H, H-14 and H-15).

Example 3 Production of Maleimido-alanine-biotin-hydrazide (MABH) (Compound of formula I with R¹ = -CH (CH₃) -)

5.0 mg (0.020 mmol) biotin hydrazide and 6.3 mg (0.023 mmol) Compound of formula II with R¹ = -CH (CH₃) - and Succinimidyloxy as the activating group was in 1 ml dry dimethylformamide dissolved under heating and worked according to Example 1. The MABH obtained (2 mg) was used for the biotinylation of Thiol groups used.

Example 4 Coupling of gamma-maleimido-butyryl-biotin hydrazide (gamma-MBBH) to antibodies

a) The S-S bonds of the "hinge" region were in themselves known manner (Arch. Biochem. Biophys. (1961), 93, 460-462) using mercaptoethylamine to form thiol groups reduced. 0.5 mg (3.125 nmol) of the reduced antibody were in 240 ul 0.1 mol / l phosphate / 5 mmol / l EDTA (ethylenediaminetetraacetate) buffer pH 6.0, which degassed and gassed with argon, with a Solution of 0.053 mg (0.125 µmol = 40 equiv.) Gamma-MBBH (Compound of formula I with R¹ = -CH₂-CH₂-CH₂-) in Dimethylformamide (10 ul) added and 1 h at + 37 ° C. incubated under argon. The conjugate formed was after gel filtration via Biogel P6 in phosphate-buffered Saline solution pH 7.2 in purified form isolated.

b) In analogy to Example 4a), 0.2 mg (1.25 nmol) of the reduced antibody in 110 µl buffer with a Solution of 0.025 mg (58.8 nmol = 47 equil.) Gamma-MBBH  implemented and the conjugate also in an analogous manner isolated.

Example 5 Coupling of β-maleimido-propionyl-biotin hydrazide (β-MPBH) to antibodies

a) The reaction was carried out in analogy to Example 4a) 40 equiv. β-MPBH (compound of formula I with R¹ = -CH₂-CH₂-).

b) The reaction was carried out in analogy to Example 4b) 120 equivl. β-MPBH.

Example 6 Coupling of maleimido-alanine-biotin-hydrazide (MABH) antibody

a) The reaction was carried out in analogy to Example 4a) 40 equiv. MABH (compound of formula I with R¹ = -CH (CH₃) -).

b) The reaction was carried out in analogy to Example 4b) 120 equiv. MABH.

Example 7 Production of Fab′-fragments of polyclonal antibodies from the rabbit

Anti-HCG antibodies from rabbits were found in themselves known manner (Arch. Biochem. Biophys. (1960), 89, 230- 244) cleaved with pepsin. F (from ') ₂ was via gel filtration isolated on Metrogel ACA 44 (LKB) and in itself  known manner (Arch. Biochem. Biophys. (1961) 93, 460-462) by reaction with 2-mercaptoethylamine in Fab ' converted.

Example 8 Production of Fab 'fragments of monoclanar antibodies from the mouse

A monoclonal antibody directed against peroxidase from the mouse became Fab 'in analogy to Example 7 worked up.

Example 9 Coupling of gamma-maleimido-butyryl-biotin hydrazide (gamma-MBBH) to Fab '

a) 0.5 mg (11 nmol) Fab 'were in 170 ul 0.1 mol / l Phosphate / 5 mmol / l EDTA (ethylenediaminetetraacetate) - Buffer pH 6.0, which degasses and gasses with argon with a solution of 0.053 mg (0.125 µmol = 11.5 equiv.) Gamma-MBBH (compound of the formula I with R¹ = -CH₂-CH₂-CH₂-) in dimethylformamide (10 µl) added and incubated for 1 h at + 37 ° C under argon. The Conjugate formed was over gel filtration Biogel P6 in phosphate buffered saline pH 7.2 isolated in purified form.

b) In analogy to Example 9a), 0.2 mg (4.3 nmol) Fab 'in 300 µl buffer with a solution of 0.025 mg (58.8 nmol = 13.7 equiv.) Gamma-MBBH implemented.  

Example 10 Coupling of β-maleimido-propionyl-biotin hydrazide (β-MPBH) to Fab '

a) The reaction was carried out in analogy to Example 9a) 10 equiv. β-MPBH (compound of formula I with R¹ = -CH₂-CH₂-).

b) The reaction was carried out in analogy to Example 9b) 12.5 equiv. β-MPBH.

Example 11 Coupling of maleimido-alanine-biotin-hydrazide (MABH) Fab '

a) The reaction was carried out in analogy to Example 9a) 10 equiv. MABH (compound of formula I with R¹ = -CH (CH₃) -).

b) The reaction was carried out in analogy to Example 9b) 12.5 equiv. MABH.

Example 12 Characterization of biotinyl whole antibody conjugates by HPLC

The purified biotinyl whole antibody conjugates of Examples 4a, b, 5a, b and 6a, b were on a Toyo Soda TSK 3000 SW HPLC column chromatographed and with the compared to the original antibody preparation as a control.  

In all cases the main peak corresponded to a molecular weight of 150,000, and in the biotinyl-antibody conjugates became a second, smaller molecular weight peak 45,000 observed.

The different biotinyl-antibody conjugates were used a 1- to 5-fold molar excess of avidin (MW = 67,000 for the tetramer) and the reaction by means of HPLC analyzed. Only main peaks were left in the evaluation of avidin and of substances with molecular weights found greater than 150,000, which is more real Biotinyl whole antibody conjugates confirmed.

Example 13 Characterization of biotinyl-Fab 'conjugates by HPLC

The purified biotinyl-Fab 'conjugates of Examples 9a, b, 10a, b and 11a, b were on a Toyo Soda TSK 3000 SW HPLC column chromatographed. It was one only peak corresponding to a molecular weight of 45,000 received. Was the Biotinyl-Fab 'conjugates an excess of avidin was added in each case chromatographic evaluation of the peak of biotinyl Fab 'conjugate of MW 45,000 almost completely disappeared and instead peaks became more molecular Substances observed what the presence of real biotinyl Fab 'conjugates confirmed.

Claims (1)

1. A method for producing a biotin derivative of a thiol, wherein a compound of formula I wherein
R¹ - (CH₂) _m - with m = 1 to 5 or
is a spacer of the formula III-R²- (CH₂) _n (III), wherein
n 1 to 5
R² is p-phenylene or
R¹ is a radical of formula IV, -CHR³- (IV) wherein
R³ means the side chain of alanine, serine, threonine, methionine, methionine sulfone, cysteic acid, aspartic acid or glutamic acid, in dimethylformamide or dimethylsulfoxide or in a mixture of dimethylformamide or dimethylsulfoxide with water or a buffer, with a solution of the thiol and together with a pH of 5 to 9 can react and the biotin derivative thus prepared is purified and isolated.