ES2265778B2 - NEW BIOTIN REAGENT, METHODS FOR PREPARATION AND MARKING, AND APPLICATIONS. - Google Patents

Abstract

New biotin reagent, methods for preparation and labeling, and applications. The present invention presents a new biotin reagent, biotin chloride or 5- ((3aS, 4S, 6aS) -2-oxo-hexahydro-1H-thieno [3,4-d] imidazol-4-yl) chloride Pentanoyl, its preparation in a single stage, as well as its labeling also in a single stage. In addition, both biotin chloride and the biotinylated marker can bind to a large number of biomolecules (proteins, enzymes, peptides, oligosaccharides or lipids), which allows their use in studies to analyze the implication of these biomolecules in biological processes as well as their use in the preparation of diagnostic systems.

Description

New biotin reagent, methods for its preparation and marking, and applications.

Object of the invention

The invention falls within the area of the Organic Chemistry and Biochemistry with application in the sectors of Biotechnology and Biomedicine. More specifically it refers to development of a new biotin reagent and its use in the preparation of labeled derivatives as well as their binding to biomolecules

State of the art

Biotin (vitamin H) is a soluble molecule in water found at low concentrations in the blood and in the tissues. Its biological function is to act as a molecule transporter of the carboxyl group. Biotin is bound covalently to the enzyme pyruvate carboxylase. A carboxyl group activated, derived from a bicarbonate ion, binds to biotin at through a reaction that requires a supply of energy from of the hydrolysis of an ATP molecule. Next this group carboxyl is transferred to the pyruvate methyl group forming oxaloacetate

Biotin also binds with a high affinity to avidin, 63 Kda glycoprotein, and other related proteins such as streptavidin, non-glycosidated protein ( K d = 10-15 M, Green NM. Adv. Prot. Chem .1975; 29, 85-133.). The affinity between avidin and biotin has a large number of applications, for example, the avidin-biotin complex has been used as a detection system where the target molecule is combined with biotin through its terminal carboxyl group, thus the molecules Biotinylated can be easily detected and separated from the solution. The biotinylation of the target molecule can be carried out without changing the biological or physicochemical properties of the target molecule and without affecting the binding capacity of biotin to avidin.

The strong interaction between streptavidin (or avidin) and biotin (Gitlin, G .; Bayer, EA and Wilchek, M. 1987, Biochem. J. 242, 923-926, Gitlin, G .; Bayer, EA and Wilchek, M. 1988, Biochem. J. 250, 291-294) is well known, in fact it is one of the strongest, within non-covalent biological interactions.

The binding takes place rapidly and is stable in a wide range of pH, temperature and denaturing conditions, which has allowed the development of a wide and diverse number of applications using streptavidin-biotin or avidin-biotin technology. (Savage et al ., Avidin-Biotin Chemistry: A Handbook, 1992: 1-23, Rockford, Pierce Chemical Company).

Biotin-streptavidin affinity is one of the most used in molecular, immunological and cellular assays. Streptavidin can be detected and quantified with a high degree of sensitivity in the complex formed, for example by marking it with an enzyme or a fluorescent or radioactive label. Streptavidin labeling has also been used to detect proteins on the cell surface, to visualize and quantify blots and carry out ELISA tests (P. Vincent, Journal of Immunological Methods , 165; 177-182, 1993).

Streptavidin can also be immobilized. on a surface so as to capture molecules or cells biotinylated. These surfaces can be used to detect the molecules of interest from complex mixtures. Therefore the streptavidin-biotin interaction has been widely used in large number of separation processes, purification and isolation, such as by chromatography of affinity, etc. This same approach has been used to lead to  conducted studies of interactions between biomolecules by use of biosensors based on plasmon resonance of surface (SPR).

The immobilization of oligonucleotides and nucleic acids is frequently used in many molecular biology procedures and in many other techniques such as sequencing, amplification, cDNA preparation and nucleic acid purification. This methodology has been adapted for use in solid phase, using a support covered with streptavidin. Streptavidin-biotin magnetic microparticles have also been prepared for use in PCR (polymerase chain reaction) (Hultman et al ., Nucleic Acids Res . 17: 4937-4946, 1989). This method is of great interest, results in good yields and is easily automated compared to traditional methods of purification based on precipitation and centrifugation.

Most applications are based on the irreversible streptavidin-biotin binding, however in those cases where dissociation of the complex is necessary it is also possible to carry it out and recover biotinylated molecules or cells in this way (Lee et al ., Anal Biochem . 206: 206-207, 1992, Elgar et al ., DNA Sequence 2: 219-226, 1992, and Conrad et al ., Nucleic Acids Res . 20: 6423-6424, 1992 and Tong et al . , Anal. Chem . 64: 2672-2677, 1992).

Biotin is a non-heterocyclic compound. aromatic with a 4-carboxybutyl tail. In the derivatives biotin binds through the 4-carboxybutyl to a connector (spacer) that It can be associated with a biomolecule (protein, carbohydrates, DNA, lipid, drug, etc.). All procedures described for carry out the reaction between biotin and the amino group of the molecule under study used the adjuvants typical of the peptide bond formation such as NHS and EDC, activators of amino and carboxyl groups respectively. Yields described in the literature for this process are low (30%) and the tedious procedure.

On the other hand, in order to use these molecules in the study of biological processes, it is important to mark these molecules so that they can be monitored. The simplest and fastest method of marking biomolecules is the introduction of a fluorophore. One of the fluorescent markers used in the literature is 2,6-diaminopyridine, in whose structure, the presence of the two amino groups allows on the one hand their binding to the conveniently functionalized biomolecule, and on the other hand their subsequent binding to a molecule of biotin, which binds strongly and selectively to avidin. Toomre et al. ( Glycobiology , 8; 653-663, 1994) use biotin and 2,6-diaminopyridine with adjuvants typical of peptide bond formation such as NHS and EDC. The yields described in the literature for this process are 31% (scheme 1).

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Scheme one

Formation of a glycoconjugate fluorescent with ring opening oligosaccharide

4

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Description of the invention

New biotin reagent, methods for its preparation and marking, and applications.

One aspect of the present invention relates to a new biotin reagent, biotin chloride or 5- ((3a S , 4 S , 6a S ) -2-oxo-hexahydro-1 H -thieno chloride [3,4] -d] imidazol-4-yl) pentanoyl.

Also, another aspect of the present invention refers to the method for the preparation of this reagent of biotin that allows your reaction quickly and easily with any properly functionalized biomolecule, as well as its Marking with a marker.

The method of preparing biotin chloride that is described is easy to make and, in addition, with it They obtain high yields, high reactivity and great application. The Preparation of biotin chloride is a simple procedure that It involves a single stage consisting of the addition of thionyl to biotin. A solid immediately precipitates, which is the reaction product that is easily isolated, for example by A simple vacuum filtration. The obtained yield is of 75%

Another aspect contemplated by the invention is the biotin chloride labeling. Marking can be done with a fluorescent marker or a radioactive marker. Marking fluorescent consists of a simple procedure that involves a single stage, that is the addition of the fluorophore to a solution of the Biotin chloride in the appropriate solvent (10% DMF (dimethylformamide) or DMSO (dimethylsulfoxide) and 90% solvent in the that the fluorophore is soluble). Among fluorophores you can use 2,6-diaminopyridine, marker fluorescent that absorbs UV-Visible. After the necessary time, 6 hours, a precipitate forms which is obtained by simple methods such as filtering. Be they obtain 100% yields.

Both the new biotin reagent and the biotinylated marker can be attached to a large number of biomolecules (proteins, enzymes, peptides, oligosaccharides or lipids) which allows its use in studies to analyze its involvement in biological processes or in the preparation of systems Diagnostic This process can be carried out in a way simple and consistent in a single stage, that is the addition of the biomolecule to a solution of biotin chloride in the solvent  appropriate (10% DMF (dimethylformamide) or DMSO (dimethyl sulfoxide) and 90% solvent in which the biomolecule is soluble). Are obtained 95% yields.

Brief description of the figures

Figure 1. Fluorescent labeling of chloride biotin (2) with 2,6-diaminopyridine (3). Through the addition of thionyl chloride (SOCl2) to biotin (1) is obtains biotin chloride (2) that is marked when added 2,6-diaminopyridine (3) obtaining the marker biotinylated (4) with a 100% yield.

Figure 2. Preparation of a biomolecule biotinylated (6) using the new biotin chloride (2). By adding the biomolecule (5) to a solution of biotin chloride (2) the biotinylated molecule (6) is obtained with 95% yield.

\ ding {226} How to carry out the invention

The present invention is further illustrated. through the following examples, which are not limiting of its scope, which is defined exclusively by the note attached claim.

Example 1 Preparation of biotin chloride

To 1 g (4.09 mmol) of biotin, in a flask of round bottom provided with a magnetic stirring core, it will be they added 8 ml (109.7 mmol) of thionyl chloride. Dissolution of the solid was immediate. Within a few seconds the chloride precipitated of acid in the form of white solid. The solid was filtered under vacuum. Be they removed the residues of thionyl chloride with high vacuum and the solid was washed with acetone. 75% yield.

Analysis calculated for C 10 H 15 ClN 2 O 2 S: C: 45.71%; H: 5.75%; N: 10.66%; S: 12.20%.

Found: C: 40.45%; H: 5.44%; N: 9.31%; S: 10.74%

MS: m / z 78 (100), 63 (92), 45 (16), 184 (10), 112 (8), 85 (7), 97 (7), 144 (6), 166 (6), 226 (2), 244 (2), 60 (48).

IR: 3250, 1702, 1653, 1481, 1319, 1271.

1 H-NMR (500 MHz, DMSO): 6.44 (sa, 2H, -NH); 4.31 (dd, 1H, J = 4.5 and 7.7 Hz, H-1 '); 4.13 (dd, 1H, J = 4.4 and 7.8 Hz, H-5 '); 3.42 (ddd, 1H, J = 1.7, 2.8 and 6.0 Hz, H-6 '); 2.80 (dd, 1H, J = 5.0 and 12.5 Hz, H-8 '?); 2.57 (d, 1H, J = 12.4 Hz, H-8 '?); 2.18 (t, 2H, J = 7.3 Hz, H-2); 1.76 (m, 1H, H-5 '?); 1.64 (m, 3H, H-4 and H-5α); 1.49 (m, 2H, H-3).

13 C-NMR (125 MHz, DMSO): 174.78 (COOH), 163.55 (C-3 '), 68.25 (C-5 '), 66.93 (C-1'), 60.64 (C-6 '), 44.90 (C-2), 38.90 (C-8 '), 33.49 (C-3), 33.18 (C-4), 29.77 (C-5).

Example 2 Biotin Chloride Fluorescent Marking

In a round bottom flask and provided with a stirring core, 0.3 g (2.7 mmol) of 2,6-diaminopyridine and 1 ml (7.2 mmol) of Triethylamine dissolved in 10 ml of dry dichloromethane. The mixture is stirred until dissolved. To this solution a slowly added solution formed by 0.35 g (1.33 mmol) of biotin chloride in 15 ml of dry dichloromethane and 2 ml of dimethylsulfoxide. It stirred for 6 h and a white precipitate was obtained, filtered under vacuum and washed with acetone. 100% yield.

Analysis calculated for C 15 H 21 N 5 O 2 S: C: 53.71%; H: 6.31%; N: 20.88%; S: 9.56%.

Found: C: 53.74%; H: 6.28%; N: 20.84%; S: 9.56%.

IR (nu in cm -1): 3407, 3275, 1684, 1659, 1641, 1459, 1399.

MS: m / z 63 (100), 78 (71), 45 (20), 109 (20), 97 (16), 85 (10), 184 (10), 244 (2).

1 H-NMR (500 MHz, d 6 -DMSO): 6.99 (t, 1H, J = 7.8 Hz, H-4 '); 6.44 (s, 1H, NH2); 6.36 (s, 1H, NH2); 5.58 (d, 2H, J = 7.8 Hz, H-3 'and H-5'); 5.31 (sa, 3H, -NH, H-2 '' and H-4 ''); 4.29 (dd, 1H, J = 5.1, J = 7.6 Hz, H-1 ''); 4.11 (dddd, 1H, J = 1.7, J = 3.3, J = 5.5, J = 7.7 Hz, H-5``); 3.43 (dddd, 1H, J = 1.7, J = 3.3, J = 5.5, J = 7.7 Hz, H-5``); 2.80 (dd, 1H, J = 5.0, J = 12.4 Hz, H-8 ''?; 2.56 (d, 1H, J = 12.4 Hz, H-8 '' \ alpha); 2.18 (t, 2H, J = 7.3 Hz, H-2); 1.76 (m, 1H, H-5 '?); 1.64 (m, 3H, H-4 and H-5α); 1.49 (m, 2H, H-3).

13 C-NMR (125 MHz, d 6 -DMSO): 180.21 (C-1), 168.30 (C-3 ''), 164.22 (C-6 'and C- 2 '), 143.85 (C-4'), 100.71 (C-3 'and C-5'), 66.62 (C-5 ''), 65.81 (C-1 '') , 64.74 (C-6``), 60.99 (C-8 '') 39.08 (C-2), 33.70 (C-3), 33.62 (C-4), 30 , 13 (C-5).

Example 3 Preparation of biotinylated biomolecule with chloride biotin

In a round bottom flask with a stirring core, 50 mg (0.16 mmol) of p -anilin- N- acetylglucosaminopyranoside and 1 mL of triethylamine dissolved in 10 mL of water were placed. The mixture was stirred until dissolved. To this solution, a solution consisting of 42 mg (0.16 mmol) of biotin chloride in 1 mL N, N- dimethylformamide was slowly added. Stirring was continued for 72 h. After this time the solvent was removed under high vacuum. 95% yield.

Analysis calculated for C 15 H 21 N 5 O 2 S: C: 53.52%; H: 6.36%; N: 10.40%; S: 5.95%.

Found: C: 53.62%; H: 6.24%; N: 10.84%; S: 5.56%.

IR (nu in cm -1): 3297, 3051, 2934, 2847, 1701, 1650.

1 H-NMR (500 MHz, d 6 -DMSO): 10.20 (s, 1H, Ar- NH -CO); 8.49 (s, 3H, OH); 8.20 (d, 1H, J = 1.8 Hz, N H -CO-CH 3); 7.45 (d, 2H, J = 9.1 Hz, H-3 '' and H-5 ''); 6.87 (d, 2H, J = 9.1 Hz, H-2 '' and H-6 ''), 6.45 (s, 1H, H-3 '), 6.39 (s, 1H, H-1 '), 4.9 (1H, d, J = 8.5 Hz, H-2'''); 4.31 (dd, 1H, J = 5.4, J = 7.4 Hz, H-6'a); 4.13 (dddd, 1H, J = 1.6, J = 1.9, J = 2.8,
J = 7.6 Hz, H-3'a); 3.67 (dd, 1H, J = 1.8, J = 10.3 Hz, H-3 '''); 3.41 (dd, 2H, J = 5, J = 12.4 Hz, H-4 '''and5'''); 3.22 (m, 3H, H-6 '''and OH-C H2 ); 2.89 (dd, 1H, J = 5.1, J = 12.4 Hz, H-6 '?; 2.75 (d, 1H, J = 12.4 Hz, H-6'?; 2.56 (ddd, 1H, J = 1.7, J = 2.7, J = 6 Hz, H-4 '); 2.2 (t, 2H, J = 7.4 Hz, H-2) ; 2.15 (s, 3H, NHCOC H 3} {), 1.76 (m, 1H, H-5), 1.64 (m, 3H, H-4 and H-5); 1.49 ( m, 2H, H-3).

13 C-NMR (125 MHz, d 6 -DMSO): 175.0 (C-1), 169.9 (NH C O-CH 3), 165.2 (C-2 '), 163.0 (C-4''), 135.3 (C-1'), 125.7 (C-2 '' and C-6 ''); 120.5 (C-3 '' and C-5 ''); 105.1 (C-2``); 82.4 (C-6 ''), 78.3 (C-4 '''), 76.4 (C-5'''); 67.2 (C-3'a); 65.3 (C-6'a); 66.2 (C-4 '); 61.0 (C-6 '); 59.3 (C-3``); 40.1 (C-2), 39.2 ( C H 2 OH); 31.0 (C-3), 28.6 (C-4), 28.1 (C-5); 25.0 (NHCO C H 3).

Claims (19)

1. Biotin reagent characterized by being biotin chloride or 5 - ((3a S , 4 S , 6a S ) -2-oxo-hexahydro-1 H -thieno [3,4-d] imidazol-4-yl chloride ) pentanoyl. 2. Method of preparing biotin chloride, which includes the following steps:

to)

addition of thionyl chloride to biotin

b)

precipitate of chloride biotin

C)

obtaining solid precipitate

d)

removal of chloride debris from thionyl

and)

solid wash.

3. Method of preparing biotin chloride according to claim 2, characterized in that the obtaining of the precipitated solid is carried out by vacuum filtration. 4. Method of preparing biotin chloride according to claim 2, characterized in that the removal of thionyl chloride residues is carried out by high vacuum. 5. Method of preparing biotin chloride according to claim 2, characterized in that the washing of the solid is carried out with acetone. 6. Biotin chloride labeling method that It comprises the following steps:

to)

marker dissolution

b)

adding the marker to the chloride of biotin in the presence of the appropriate solvent

C)

obtaining the precipitate

d)

washing of the precipitate.

7. Biotin chloride labeling method according to claim 6, characterized in that the solvent used is 10% dimethylformamide or dimethylsulfoxide and 90% solvent in which the label is soluble. 8. Biotin chloride labeling method according to claim 6, characterized in that the labeling is carried out with a fluorophore. 9. Biotin chloride labeling method according to claim 8, characterized in that the fluorophore used is 2,6-diaminopyridine. 10. Biotin chloride labeling method according to claim 6, characterized in that the stirring is carried out for 6 hours. 11. Biotin chloride labeling method according to claim 6, characterized in that the obtaining of the precipitated solid is carried out by vacuum filtration. 12. Method of preparing biotin chloride according to claim 6, characterized in that the washing of the solid is carried out with acetone. 13. Molecule preparation method biotinylated comprising the following steps:

to)

dissolution of the biomolecule

b)

addition of the biomolecule to the chloride of biotin in the presence of the appropriate solvent

C)

obtaining the precipitate.

14. Method for preparing biotinylated molecules according to claim 13, characterized in that the solvent used is 10% dimethylformamide or dimethylsulfoxide and 90% solvent in which the biomolecule is soluble.

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15. Method of preparing biotinylated molecules according to claim 13, characterized in that the obtaining of the precipitate is carried out by filtration. 16. Method of preparing biotinylated molecules according to claim 13, characterized in that the biomolecules are proteins, enzymes, peptides, oligosaccharides or lipids. 17. Method for preparing biotinylated molecules according to claim 13, characterized in that the biotin chloride is labeled. 18. Use of biotin chloride, according to previous claims, in the study of the implication of biomolecules or analogues thereof in biological processes. 19. Use of biotin chloride, according to claims 1 to 17, for the development of systems of diagnosis.