WO2007017540A1 - Novel biotin reagent, methods of preparing and labelling same and uses thereof - Google Patents

Abstract

The invention relates to a novel biotin reagent, methods of preparing and labelling same and uses thereof. More specifically, the invention relates to a novel reagent of biotin, biotin chloride or chloride of 5-((3aS,4S,6aS)-2-oxo-hexahydro-1H-tieno[3,4-d]imidazole-4-yl)pentanoyl, as well as the one-step preparation and the one-step labelling of same. Both the biotin chloride and the biotinylate marker can be bound to a large number of biomolecules (proteins, enzymes, peptides, oligosaccharides or lipids), thereby enabling the use of same in studies analysing the involvement of said biomolecules in biological processes and 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 Organic Chemistry and Biochemistry with application in the Biotechnology and Biomedicine sectors. More specifically, it refers to the development of a new biotin reagent and its use in the preparation of labeled derivatives as well as its binding to biomolecules.

STATE OF THE TECHNIQUE:

Biotin (vitamin H) is a water soluble molecule that is found at low concentrations in the blood and tissues. Its biological function is to act as a transport molecule of the carboxyl group. Biotin is covalently bound to the enzyme pyruvate carboxylase. An activated carboxyl group, derived from a bicarbonate ion, is coupled to biotin through a reaction that requires a supply of energy from the hydrolysis of an ATP molecule. This carboxyl group is then 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 (X¿ = 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, in this way biotinylated molecules can be easily detected and separated from the solution.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 mapping has also been used to detect proteins on the cell surface, to visualize and quantify blots and carry out ELISA-type assays (P. Vincent, Journal of Immunological Methods, \ 65; 177-182, 1993).

Streptavidin can also be immobilized on a surface so as to capture biotinylated molecules or cells. These surfaces can be used to detect the molecules of interest from complex mixtures. Therefore, the streptavidin-biotin interaction has been widely used in a large number of separation, purification and isolation processes, such as affinity chromatography, etc. This same approach has been used to carry out studies of interactions between biomolecules through the use of biosensors based on surface plasmon resonance (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 thus recover biotinylated molecules or cells (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., Α "α /. Chem. 64: 2672-2677, 1992).

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

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 easiest and fastest method of biomolecule mareaje 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) employ 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).

Scheme 1. Formation of a fluorescent glycoconjugate with opening of the oligosaccharide ring.

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 - ((3a5 ^r , 45 ^í , 6a, S) -2-oxo-hexahydro-lH ^' -thieno [3, 4- d] imidazol-4-yl) pentanoyl. Likewise, another aspect of the present invention refers to the method for the preparation of this biotin reagent that allows its reaction in a quick and simple way with any correctly functionalized biomolecule, as well as its marking with a marker.

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

Another aspect contemplated by the invention is the mapping of biotin chloride. The marking can be done with a fluorescent marker or a radioactive marker. Fluorescent marking consists of a simple procedure that involves a single stage, that is, the addition of fluorophore to a solution of biotin chloride in the appropriate solvent (10% DMF (dimethylformamide) or DMSO (dimethyl sulfoxide) and 90% solvent in which the fluorophore is soluble). Among the fluorophores, 2,6-diaminopyridine, a fluorescent marker that absorbs Ultraviolet-Visible, can be used. After the necessary time, 6 hours, a precipitate forms which is obtained by simple methods such as filtering. 100% yields are obtained.

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

Figure 1. Fluorescent marking of biotin chloride (2) with 2,6-diaminopyridine (3). By adding thionyl chloride (SOCl ₂ ) to biotin (1), biotin chloride (2) is obtained, which is marked when 2,6-diaminopyridine (3) is added, obtaining the biotinylated marker (4) with a yield of 100 %.

Figure 2. Preparation of a biotinylated biomolecule (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 in 95% yield.

> MODERATE THE INVENTION

The present invention is further illustrated by the following examples, which are not limited to its scope, which is defined exclusively by the attached claim.

Example 1. Preparation of biotin chloride

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

Analysis calculated for C ₁₀ Hi ₅ 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, IH ₃ J = 4.5 and 7.7 Hz, H-1 '); 4.13 (dd, IH, J = 4.4 and 7.8 Hz, H-5 '); 3.42 (ddd, IH, J = 1.7, 2.8 and 6.0 Hz, H- 6 '); 2.80 (dd, IH, J = 5.0 and 12.5 Hz, H-8'β); 2.57 (d, IH, J = 12.4Hz, H-8'α); 2.18 (t, 2H, J = 7.3 Hz, H-2); 1.76 (m, IH, H-5'α); 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-I '), 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. Fluorescent biotin chloride mapping

In a round bottom flask with a stirring core, 0.3 g (2.7 mmol) of 2,6-diaminopyridine and ImI (7.2 mmol) of triethylamine dissolved in 10 ml of dry dichloromethane were placed. The mixture was stirred until dissolved. To this solution, a solution consisting of 0.35 g (1.33 mmol) of biotin chloride in 15 ml of dry dichloromethane and 2 ml of dimethylsulfoxide was added slowly. It was 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 ₂ IN 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 (v 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, ^ DMSO): 6.99 (t, IH, J = 7.8 Hz, H-4 '); 6.44 (s, IH, NH ₂ );

6.36 (s, IH, NH ₂ ); 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 ₅ HI"); 4.11 (dddd, IH, J = 1.7, J = 3.3, J =

5.5, J = 7.7 Hz, H-5 "); 3.43 (dddd, IH, J = 1.7, J = 3.3, J = 5.5, J = 7.7 Hz, H-5 "); 2.80 (dd, IH, J = 5.0, J = 12.4 Hz, H-8 "β); 2.56 (d, IH, J = 12.4 Hz, H-8" α); 2.18 (t,

2H, J = 7.3 Hz, H-2); 1.76 (m, IH, H-5'α); 1.64 (m, 3H, H-4 and H-5α); 1.49 (m, 2H,

H-3). ¹³ C-NMR (125 MHz, Í / ¿-DMSO): 180.21 (CI), 168.30 (C-3 "), 164.22 (C-6 'and C-T), 143.85 ( C-4 '), 100.71 (C-3' and C-5 '), 66.62 (C-5 "), 65.81 (CI"), 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 biotin chloride.

In a round bottom flask with a stirring core 50 mg (0.16 mmol) of p-aniline-iV-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, iV-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 (v in cm ⁴ ): 3297, 3051, 2934, 2847, 1701, 1650.

¹ H-NMR (500 MHz, ^ ₆ -DMSO): 10.20 (s, IH, Ar-NH-CO); 8.49 (s, 3H, OH); 8.20 (d, IH, J = 1.8 Hz, NH-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, IH, H-3 '), 6.39 (s, IH, H- I '), 4.9 (IH, d, J = 8.5 Hz, H-2'"); 4.31 (dd, IH, J = 5.4, J = 7.4 Hz, H-6 'a); 4.13 (dddd, IH, J = 1.6, J = 1.9, J = 2.8, J = 7.6 Hz, H-3'a); 3.67 (dd, IH, J = 1.8, J = 10.3 Hz, H-3 '"); 3.41 (dd, 2H, J = 5, J = 12.4 Hz, H-4 '"and 5""); 3.22 (m, 3H, H-6 '"and OH-CH ₂ ); 2.89 (dd, IH, J = 5.1, J = 12.4 Hz, H-6'β); 2, 75 (d, IH, J = 12.4 Hz, H-6'α); 2.56 (ddd, IH, 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, NHCOCH ₃ ); 1.76 (m, IH, H-5); 1.64 ( m, 3H, H-4 and H-5); 1.49 (m, 2H, H-3).

¹³ C-NMR (125 MHz, ^ DMSO): 175.0 (CI), 169.9 (NHCO-CH ₃ ), 165.2 (C-2 '), 163.0 (C-4 "), 135 , 3 (C-I '), 125.7 (C-2 "and C-6"); 120.5 (C-3 "and C-5"); 105.1 (C-2'"); 82.4 (C-6 '") ₅ 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 (CH ₂ OH); 31.0 (C-3), 28.6 (C-4), 28.1 (C-5); 25.0 (NHCOCH ₃ ).

Claims

> CLAIMS 1. Biotin reagent characterized by being biotin chloride or 5- ((3a5,4 ₁ S ' ₅ 6aS) -2-oxo-hexahydro-lH-thieno [3,4-d] imidazol-4-yl) chloride pentanoyl 2. Method of preparation of biotin chloride, comprising the following steps: a) addition of thionyl chloride to biotin b) precipitate of biotin chloride c) obtaining the precipitated solid d) removal of thionyl chloride residues e) washing of the solid 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 mapping method comprising the following steps: a) dissolution of the marker b) addition of the marker to biotin chloride in the presence of the appropriate solvent c) obtaining the precipitate d) washing the precipitate 7. Biotin chloride chloride 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. Method of biotin chloride marking according to claim 6, characterized in that the marking is carried out with a fluorophore. 9. Biotin chloride mapping method according to claim 8, characterized in that the fluorophore used is 2,6-diaminopyridine. 10. Biotin chloride dyeing method according to claim 6, characterized in that the stirring is carried out for 6 hours. 11. Biotin chloride chloride 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. Method of preparation of biotinylated molecules comprising the following steps: a) dissolution of the biomolecule b) addition of the biomolecule to the biotin chloride 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. 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 analogs thereof in biological processes. 19. Use of biotin chloride, according to claims 1 to 17, for the development of diagnostic systems.