WO2009004106A1

WO2009004106A1 - Porous biotin carrier, and associated production methods and uses

Info

Publication number: WO2009004106A1
Application number: PCT/ES2008/070105
Authority: WO
Inventors: Francisco SANTOYO GONZÁLEZ; Fernando HERNÁNDEZ MATEO; Mariano ORTEGA MUÑOZ; Rafael Salto Gonzalez; Maria Dolores GIRÓN GONZÁLEZ; Natalia Sevillano Tripero
Original assignee: Universidad De Granada
Priority date: 2007-07-02
Filing date: 2008-05-29
Publication date: 2009-01-08
Also published as: ES2310969B1; ES2310969A1

Abstract

The invention relates to a compound comprising biotin joined to a porous carrier by means of a linker (joining or linking group) containing the 1, 2, 3-triazole group. The invention also relates to the method for obtaining said compound and to the uses thereof for purifying avidin and biotinylated proteins, and for the fluorescent marking of avidin.

Description

BIOTINE-POROUS SUPPORT, METHODS OF OBTAINING AND USES

The present invention relates to a compound comprising biotin linked to an inorganic porous support by means of a "linker" (binding group) containing the group 1,3,3-triazole. More particularly, the inorganic porous support is silica. In addition, it refers to its method of obtaining and its uses for the purification of avidin and biotinylated proteins, as well as for fluorescent avidin marking

STATE OF THE PREVIOUS TECHNIQUE

Biotin is an essential vitamin in the metabolism that constitutes the prosthetic group of a set of enzymes called carboxylases that participate in key reactions of the intermediate metabolism. Biotin deficiency, or genetic defects that prevent its covalent binding to carboxylases, leads to serious metabolic diseases, which normally produce situations of acidosis and coma.

A possible situation of biotin deficiency is caused by an excessive intake of raw eggs. In egg white there is a protein called avidin (Cf. DeLange, RJ., 1969, J. Biol. Chem., Vol. 245, pp.

907-916), which presents the ability to bind strongly and sequester biotin. In fact, the binding constant between biotin and avidin is approximately 10 ^{~ 15} M, which probably constitutes the greatest affinity for a ligand described for any biological system.

This high affinity, as well as the relative ease of obtaining avidin, has determined that there is a high interest in the study and development of affinity systems that are based on the strong interaction between these two molecules. Thus, protein, nucleic acid, various cell constituent and even whole cell mapping systems have been developed to later be recognized by avidin molecules, either immobilized (creating affinity chromatography systems) or marked by the binding to fluorophores or enzymes (creating detection systems, usually in solid phase). The techniques described in the previous paragraph have been used classically in immunology for the detection of previously biotinylated antigens or antibodies. Currently, this technology is in an expansion process, extending its use to proteomics and genomics, in the marking, separation and / or immobilization of proteins or nucleic acids.

The interaction of avidin with biotin occurs under physiological conditions of pH and ionic strength. There are numerous reagents for the biotinylation of proteins and there is commercially avidin (or an analogous protein of its bacterial origin, streptavidin) either alone, labeled with different fluorophores or immobilized on agarose.

An important problem, and only partially solved, of this technology is, paradoxically, the high affinity of avidin for biotin. This high affinity makes it necessary to use extreme conditions to break the interaction of both molecules. These extreme conditions usually involve the use of denaturing agents such as SDS or guanidinium hydrochloride together with pHs below 2.

In chromatographic techniques based on the interaction of the avidin with the biotin, either for the purification of the avidin itself or for the affinity chromatography of molecules labeled with biotin, the use of these extreme conditions of elution and the consequent denaturation of the proteins hinders subsequent applications.

To avoid this problem, the use of iminobiotin has been proposed, with a lower affinity for avidin, as a ligand. However, the use of iminobiotin is much more expensive than that of biotin in the mapping of ligands and also cannot be applied to biotinylated proteins naturally. Another alternative has been the use of affinity columns that employ monomeric avidin.

The initial purification of avidin by affinity chromatography was performed in 1968 (Cf. Cuatrecasas, P. et al., 1968, Biochem. Biophys. Res. Commun., vol. 33, pp. 235-239) using a biocitin-sepharose column. The avoidance of avidin from this column involved the use of 6 M guanidinium chloride at pH 1.5. Later, in 1980 (Cf. Hofmann et al., 1980, Proc. Nati. Acad. Sci., Vol. 77, pp. 4666-4668), the use of iminobiotin column for streptavidin purification using conditions of streptavidin was described. softer elution. The binding of avidin or streptavidin to this type of columns implies the use of a high pH (around 11) since at this pH the effective binding of the iminobiotin to the avidin occurs since the free base of the iminobiotin is Ia which is recognized by avidin (Cf. Green, NM, 1966, Biochem. J., vol 101, pp. 774-780). Elution in this type of columns occurs at pH 4.

Other proposed strategies have been the use of other biotin derivatives such as destiobiotin, whose lower affinity for binding to avidin allows its displacement through unmodified biotin (Cf. Hirsch, JD et al., 2002, Anal. Biochem., Vol 308 , pp. 343-357). In any case, iminobiotin columns are still used in the purification of avidin and the typical yield thereof is approximately 90% with a binding capacity of approximately 0.75 mg of avidin per mL of resin used (Cf. Heney, G . et al., 1981, Anal. Biochem., vol. 114, pp. 92-96). In many cases, although the purification is fundamentally based on an affinity stage, additional steps are incorporated such as a previous precipitation with ammonium sulfate or an ion exchange after affinity chromatography.

Currently there are different biotinylated supports on the market, the most widespread being those based on agarose and sepharose. Similarly, biotin and related compounds have been covalently bound to a variety of surfaces, primarily glass, silicon oxide, polymers and gold. The covalent binding of biotin to these solids or surfaces is carried out using active derivatives of biotin and supports or surfaces functionalized in addition to both chemical and photochemical methods. The majority of activated biotin derivatives use the side chain of the valeric acid to incorporate different groups that do not interfere in the union with the avidin and their counterparts Among the most used are activated esters derived from N-hidoxysuccinimide, maleimide derivative, iodoacetyl derivative, pyridyl disulfide derivative, hydrazide derivative and other derivatives containing photoactivatable groups that are anchored to the supports or surfaces through amide type junctions , thioether, disulfide or hydrazone primarily (cf. Smith, CL, et al., 2006, Top. Curr. Chem. VoI 261, pp. 63-90)

The cycloaddition of alkynes and azides (Huisgen Reaction) (Cf. R. Huisgen, In 1,3-Dipolar Cycloaddition Chemistry (Ed .: A. Padwa), Willey, New York, 1984, pp. 1-176) has recently established as an important synthetic tool within the concept of "click-chemistry" (cf. KoIb HC et al. 2001, Angew Chem lnt Ed, vol. 40, pp. 2005) especially since the discovery of its regioselective catalysis and at room temperature by Cu (I) (cf. Rostovtsev, VV et al. 2002, Angew. Chem. Int. Ed., vol. 41, pp. 2596; TornΘe, CW et al. 2002, J. Org. Chem., vol. 67, pp. 3057-3064; WO03101972 A1). The main advantages of this reaction are the easy introduction of azido and alkyne groups in the substrates and the stability of both functions that tolerate water and oxygen, and that allow the modular assembly of different molecules. The formation of the triazoles resulting from the fusion of both functions is irreversible and usually takes place with very high yields. Due to these remarkable characteristics, a large number of applications in biomedical science, organic synthesis and materials science have been developed through the use of "click-chemistry" (cf. Wang, Q. et al. 2005, Lett. Org Chem., Vol. 2, pp. 293-301; KoIb, HC et al. 2003, Drug Discov. Today, vol. 8, pp. 1128-1137; Bock, VD, et al. 2005, Eur. J. Org. Chem., Pp. 51-68; Lutz, JF 2007, Angew. Chem. Int. Ed. Vol. 46, pp. 1018-1023). In particular, the functionalization of supports based on silica gel (cf. Lummerstorfer, T. et al. 2004, J. Phys. Chem. B, vol. 108, pp. 3963-3966) and agarose (cf. Punna, S et al., 2005, Bioconjugate Chem., vol. 16, pp. 1536-1541) was performed using this methodology. EXPLANATION OF THE INVENTION

In the present invention there is provided a new compound comprising biotin linked to a porous support by means of a linker group (or "linker"). In addition to providing its method of obtaining and its uses.

By using the compound of the invention an appropriate system is provided for:

o Purification of avidin. This system is cheaper than using iminobiotin; with ease of elution of bound avidin; and with the possibility of scaling it in microfilter and centrifugation systems.

o Fluorescent avidin column marking with the following advantages: ease of operation of the marking; and possibility of performing the marking without affecting one of the binding sites of the avidin to the biotin that remains protected by the binding to the porous support through the biotin.

o Purification of biotinylated proteins in vitro and in general biotinylated ligands. Depending on the elution pH used, it is possible to select specifically the biotin-avidin binding to be released: the binding with the supported biotin or the binding with the biotin of the ligand. In addition, purified ligands can be easily characterized by MALDI-TOF spectrometry.

A first aspect of the present invention refers to the compound, hereinafter compound of the invention, which comprises: a. biotin or any of its derivatives of general formula (I); b. a "linker" or linker comprising a triazole group, more specifically a 1,3,3-triazole group; and c. a porous support.

(I) where: X is S or SO ₂ , preferably X is S; Y is O or NH, preferably Y is O;

Z is CH ₂ or CH ₃ when X does not exist; preferably Z is CH ₂ . R ¹ is O or NH, preferably R ¹ is NH; Y

R ² is a group selected from a (C1-C10) alkyl; (CH ₂ CH ₂ O) _n CH ₂ , where n has a value from 0 to 10; or a (CH ₂ ) ₄ CH (NH ₂ ) COOCH ₂ . Preferably R ² is a methylene.

The symbol ^"1 ^ indicates the point of union of the biotin or any of its derivatives with the" linker ".

By "biotin derivatives" we mean compounds that have a structure analogous to biotin and whose biological function is similar, such as biocitin, iminobitin, destiobitin, etc.

In a preferred embodiment of the compound of the present invention, the "linker" has the structure that is represented by the general formulas (II) or (III):

(H)

where:

R ³ is a group selected from a (C1-C10) alkyl; or

((CCHH ₂₂ CCHH ₂₂ OOy) _n CH ₂ , where n has a value of 0 to 10. Preferably R ³ is a methylene.

R ⁴ is selected from the group comprising O, C (O) R ¹ , R ¹ C (O) R ¹ or R ¹ C (S) R ¹ (where R ¹ is O or NH); Y

m has the value from 1 to 11. Preferably m is 1, 2 or 3.

The symbol ^ indicates, in these structures, the point of attachment of the "linker" with the biotin or any of its derivatives.

By "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 10 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl , n-pentyl, etc.

Depending on the structure of the "linker", the compounds of the invention will be represented by the following general formulas (IV) or (V):

As "porous solid support" we mean here an inorganic porous support, such as but not limited to silica, alumina, siliceous earth, zeolite, or an organic porous support selected from the group comprising celluloses or resins, as for example but not limited to polyamine or Merrifield resin. Preferably the support is an inorganic material, and more preferably the support is silica.

A preferred embodiment of the invention comprises a compound of formula (Vl), also referred to herein as "biotin-silica":

(Vl)

Another aspect of the present invention relates to a process for obtaining the compounds of the invention, which comprises the following steps: a) functionalization of the porous support using a compound comprising an azido (N ₃ ) or alkyne (C≡CH) terminal group.

b) functionalization of biotin or any of its derivatives with an alkyne (C≡CH) or azido (N ₃ ) terminal group;

c) reaction of the functionalized support of step (a) with biotin or any of its functionalized derivatives complementary to step (b) by reaction of 1, 3-dipolar cycloaddition ("click-chemistry").

This procedure can be described by the following reaction scheme, where the compound (VII) and (VIII) would be the biotin derived compounds obtained in step (b) and the compound (IX) and (X) would be the functionalized porous support with an azido or alkyne group obtained in step (a) giving rise to the compound of general formula (IV) or (V), obtained in step (c), comprising biotin, or any of its derivatives, attached to a support porous by means of a "linker" (binding group) comprising the group 1, 2,3-triazole:

where X, Y, ZR ¹ , R ² , R ³ , R ⁴ and m are described above.

The reaction that occurs between the alkyne and azido groups to give rings of 1,3,3-triazoles is a 1,3-dipolar cycloaddition which, for the particular case of these functional groups, is known in the state of the art as "click-chemistry" reaction.

The reaction of the present invention can be carried out by means of the same conditions as other click-chemistry reactions described in the state of the art, temperature, reaction time, etc. including the use of catalysts already described. In a preferred embodiment of the present invention, the catalyst used in the process for obtaining compound (I) or (II) is the 3P complex (EtO). Cu (I) (Et = ethyl).

In a preferred embodiment of the invention, the cycloaddition reaction occurs between an azido group of the functionalized porous support and an alkyne group of the functionalized biotin.

Another aspect of the present invention relates to the use of the compounds of the invention for the purification of avidin.

Another aspect of the present invention relates to the use of the compounds described in the present invention for the immobilization of avidin.

Upon immobilization of the avidin in the compound of the invention, a complex is formed which can subsequently be used for the purification of biotinylated ligands or for the avidin labeling.

Therefore, another aspect of the present invention relates to this avidin-compound complex of the invention.

The purification of biotinylated ligands is one of the main applications pursued with the use of this technology based on the interaction of avidin with biotin, which is the identification and isolation of biotinylated proteins in vitro. With the current development of proteomics, the ability to identify and purify the proteins thus modified, using mild conditions that allow their subsequent analysis by two-dimensional electrophoresis and mass spectrometry, is a pressing need.

The purification of avidin and / or biotinylated ligands using the avidin-compound complex of the invention can be carried out by means of adequate pH selection. Thus, for example, the biotinylated avidin-ligand complex can elute at pH <2.5, while at pH 4 it elutes only the biotinylated ligand.

A further aspect of the present invention refers to the use of the compound of the invention for fluorescent avidin marking once it has been previously immobilized to said compound of the invention. This operation allows the safeguarding of the active center of the avidin through which the avidin has been bound to the compound of the invention, thus maintaining its functionality once it is eluted from the support.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1.- Shows the affinity chromatography of the avidin using the compound of formula (Vl) (also called biotin-silica). A.- Chromatogram of the purification of avidin from a raw egg extract B.- Electrophoresis in SDS-PAGE of the fractions charged and eluted in the column.

FIG. 2.- Shows an SDS-PAGE gel of the eluted fractions for the biotin-silica compound.

FIG. 3.- Shows the avidin mapping with compound 7 (2-hydroxyethoxy) -4-nitro-2,1,3-benzooxadiazole (NBD-OCH ₂ CH ₂ OH). A.- Chromatogram of the elution of the biotin-silica compound. B.- SDS-PAGE electrophoresis of the eluted fractions. Where "L" corresponds to the washing of the column prior to elution. C- Spectrum emission of fractions corresponding to the wash and the two eluted peaks. The excitation was carried out at 460 nm.

FIG. 4.- Shows the purification of biotinylated horseradish peroxidase in vitro A.- Chromatogram of the elution in the resin. The absorbance at 440 nm is an index of the enzymatic activity of the eluted POD. B.- SDS-PAGE electrophoresis of the eluted fractions. POD and avidin have been included as electrophoresis standards.

DETAILED EXHIBITION OF REALIZATION MODES

Next, the invention will be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the compounds of the invention. EXAMPLE 1.- Preparation of the biotin-silica compound

The synthesis of biotin-silica was carried out in several steps:

a) Preparation of 3-azidopropyl-triethoxysilane (Xl).

To a solution of 3-chloropropyl-triethoxysilane (2.31 g, 9.6 mmol) and tetrabutylammonium iodide (0.02 g, 0.05 mmol) in butanone (25 ml_), sodium azide (3.120 g, 48 mmol) was added and the mixture was heated reaction at reflux for 50 hours. After this time, it was filtered over celite and the solvent was evaporated in vacuo. The reaction crude was dissolved in dichloromethane (150 ml_) and washed with water (2 x 20 ml_). The organic layer was dried (Na2SO ₄ ) and evaporated to give the compound of formula (Xl) (1.9 g) as a sirupous substance that was directly used without another purification process.

(Xl)

b) Preparation of azido-silica (XII): Silica gel (4g) was suspended in toluene (20 mL) and 3- azidopropyl-triethoxysilane (Xl) (1 g) was added. The reaction mixture was heated at reflux for 2 h; Approximately half of the volume was evaporated to remove the ethanol formed, the volume removed from toluene was added and heated at reflux for 1 h. It filtered the reaction mixture, washed with dichloromethane (4 x 50 mL) and dried under vacuum (1 mm Hg) at 50 ⁰ C for 16h.

c) Preparation of propargilamide-biotin (5 - [(4f?) - 2-oxohexahldro-

1 / - / - thieno [3,4-d] imidazol-4-yl] -Λ / -prop-2-inylpentanamide) (XIII).

Biotin (370 mg) was dissolved in distilled thionyl chloride (5 ml_) and stirred for 30 min. The thionyl chloride was evaporated in vacuo and coevaporated with anhydrous toluene. Biotin acid chloride was dissolved in anhydrous CI ₂ CH ₂ (5 ml_) and an excess of propargyl amine (207 μl_, 3 equiv.) Was added. The reaction mixture was stirred under argon for 30 min. and after this time the solvent was evaporated and purified by column chromatography (CI ₂ CH ₂ : Me0H, 9: 1) to obtain a white solid corresponding to (XIII) (359 mg, 84%).

"a" represents: i) CI ₂ SO (5ml_), t. amb., 30 min ii) NH ₂ CH ₂ C≡CH, CH ₂ CI ₂ .

d) Binding of propargilamide-biotin (XIII) to azido-silica (XIV). In a solution of the biotin-derived alkyne (XIII) (300 mg, 1.06 mmol) in dry DMF (10 mL), the azido-silica (XII) (3 g) was suspended and the catalyst (EtO ^ P CuI (10 % in mmol, 37 mg) The reaction was irradiated at 800W and 8O ⁰ C for 1 h in a Milestone Star Microwave Labstation until the IR spectrum or thin plate chromatography showed the total disappearance of the biotin. resulting biotin-silica (Vl) and washed with MeOH (2 x 30 mL), EDTA disodium salt (50 mM, 2 x 30 mL), water (2 x 30 mL), acetone (2 x 30 mL) and CH ₂ CI ₂ (2 x 30 mL). biotin-silica (Vl) is then dried in vacuo (1 mm Hg) at 50 ⁰ C for 16 h yielding 3.240 g-

EXAMPLE 2.- Purification of egg white avidin. The ability to bind avidin to biotin-silica (Vl) from egg white samples was tested. For this, column chromatography systems were used as well as rapid microfiltration centrifugation systems. This last system allowed the simple handling of numerous small volume samples in a reduced time since it requires small centrifugation times of approximately 30 seconds. In both cases, avidin was purified from egg white previously sonicated using methods previously described previously (Cf. Hofmann et al., 1980, Proc. Nati. Acad. Sci., Vol. 77, pp. 4666-4668; Airenne et al., 1997, Protein expression and purification, vol. 9, pp. 100-108; Hytonen et al, 2003, Biochem. J., vol 372, pp. 219-225).

In FIG. 1 shows the results of an affinity chromatography on a biotin-silica (Vl) column. A diluted 1: 1 sample of egg white previously sonicated at pH 11 was loaded on said column. A wash was performed with a buffer at pH 11 and subsequently two sequential elutions with a carbonate buffer at pH 4 and 0.1 M glycolol (pH = 2.5).

The pH was monitored as well as the absorbance at 280 nm and an electrophoresis was performed in 15% SDS-PAGE of the eluted fractions. During the whole process the temperature was maintained at 4 ⁰ C. It could be observed that the column efficiently retains a high percentage of the avidin content of the crude extract, which was subsequently eluted almost quantitatively at pH 2.5. On the contrary, elution at pH 4 was not able to break the binding of avidin to the matrix. The approximate yield of the process is 0.1 mg of avidin per gram of resin.

In other experiments 200 mg of functionalized silica which were incubated at 4 ⁰ C for one hour with ml_ crude extract from egg to pH 11. Subsequently the suspension resin and crude extract used was placed in a microfilter and centrifuged for 30 seconds. The resin was washed with buffer at pH 11 and subsequently eluted with buffers containing biotin (10 mM) of pH 4 and pH 2.5. An SDS-PAGE electrophoresis of the eluted fractions is shown in FIG. 2. Could Observe how using this technique it is possible to bind avidin to the column that is specifically eluted at pH 2.5.

The data obtained indicate that with the biotin-silica (Vl) an unexpected result is obtained and with a high applicability: the elution of the avidin bound to the column under relatively mild conditions, avoiding the use of chaotropic agents or detergents. This allows the development of biotin columns based on this matrix instead of the classic iminobiotin columns, which translates into a considerable reduction in the cost of this technology. Additionally, it should be noted that the results of the electrophoresis of the purified fractions obtained a high degree of purity (> 96% measured by protein electrophoresis), which allows purification in a single step.

In addition, alternatively, the avidin can be charged at pH 7.5 and likewise the avidin can be eluted in a 0.2 N HCI solution that can subsequently be neutralized using volatile buffers (for example carbonate / bicarbonate buffer) which allows obtaining the protein in Very low ionic strength conditions.

EXAMPLE 3.- Fluorescent dyeing of egg white avidin bound to biotin-silica resin (Vl).

Since the biotin-silica resin allowed the purification of avidin under relatively mild conditions, the column-based dyeing of the avidin bound to the resin was proceeded with a fluorophore. To do this, compound 7 (2-hydroxyethoxy) -4-nitro-2,1,3-benzooxadiazole (NBD-OCH ₂ CH ₂ OH) was used.

For the avidin mapping, the biotin-silica resin (2 g) was used, which was incubated with 15 ml_ of a raw sonicized egg extract diluted 1: 1. The pH was set at 11 and after one hour of incubation, the suspension of the resin was compacted in a column. After washing, 2 ml_ of a 2 mM solution of NBD-OCH ₂ CH ₂ OH at pH 11 were added (in all cases this washing solution at pH 11 is formed by 50 mM Na ₂ CO ₃ , 1 M NaCI pH 11) and the solution was recirculated for 16 hours through the resin at room temperature. The column was then washed with buffer at pH 11 and the bound avidin was eluted with a 0.2 N solution of HCI. The results obtained are shown in FIG. 3 and demonstrate that it is possible to perform the avidin-linked biotin-silica binding with said reagent NBD-OCH2CH2OH. The avidin mareaje with this reagent made the elution of the column at pH 1 was faster causing two peaks. A first elution peak with a higher protein fluorescence ratio and a second peak with a lower tidal ratio and greater retention in the column. This tide ratio was confirmed by MALDI-TOF. An additional screening test was obtained by observing the fluorescence spectrum of the labeled avidin and comparing it to that of the free compound. As expected, there was a slight shift in the maximum emission that was due to protein binding.

EXAMPLE 4.- Purification of biotinylated proteins in vitro by avidin bound to the biotin-silica resin (Vl).

To evaluate the capacity of the biotin-silica resin for the purification and characterization of biotinylated proteins in vitro, two experiments were performed.

1 ^o A green fluorescent protein (GFP) was labeled with biotin using the succinyl ester of 6-biotinyl hexanoic acid. Subsequently, it was purified by binding to avidin that had previously been immobilized on the biotin-silica using the microfilter system with only 100 mg of silica-biotin resin. Elution was performed with 0.2 N HCI and the eluate, after being lyophilized, analyzed by MALDI-TOF. Corresponding molecular weights were observed with the avidin monomer, as well as that of a GFP molecule modified with 4 biotins. Thus, it was demonstrated that this technique is suitable for the purification and characterization of biotinylated proteins. 2nd The horseradish peroxidase was labeled with the succinyl ester of 6-biotinyl hexanoic acid. The labeled peroxidase was separated from the biotinylating agent by chromatography on sephadex G-50 and was subsequently used to check the immobilization capacity on the biotin-silica resin. This resin, previously loaded with avidin, was eluted after the binding of biotin-peroxidase at pH 4 and pH 2. Elution of the peroxidase bound to the column was monitored enzymatically using the Trinder reagent. The results obtained are shown in FIG. 4. It could be observed that the biotinylated peroxidase was recognized by the avidin attached to the column and can be eluted in a selective manner at pH 4 (the elution of the peroxidase precedes a fraction to the change of the pH in the chromatography). This elution was produced without releasing the avidin bound to the column (see the electrophoresis in SDS-PAGE fraction 5) that is only eluted at pH 2 (Fraction 11 in the electrophoresis in SDS-PAGE).

Therefore, this system has the advantage of allowing the elution of the avidin-biotinylated ligand complex if an HCI buffer at pH 1-2.5 is chosen as the elution system or the specific elution of the biotinylated ligand if an elution system is chosen as a buffer system at pH 4.

Claims

1. Compound comprising: a. biotin or any of its derivatives of general formula (I); b. a "linker" or linker comprising the group 1,3,3-triazole; and c. a porous support.

(I where:

X is S, the group SO ₂ Ó does not exist;

Y is O or NH;

Z is CH ₂ or CH ₃ when X does not exist. R ¹ is 0 or NH; Y

R ² is a group selected from a C ₁ -C ₁ ₀ alkyl, (CH ₂ CH ₂ O) _n CH ₂ , where n has a value of 0 to 10, or (CH ₂ ) ₄ CH (NH ₂ ) COOCH ₂ .

2. Compound according to claim 1, wherein X is S.

3. Compound according to any of claims 1 or 2, wherein Y is O.

4. Compound according to any of claims 1 to 3, wherein R ¹ is NH.

5. Compound according to any of claims 1 to 4, wherein R ² is CH ₂ .

6. Compound according to any of claims 1 to 5, wherein the binder has the general formula (II) or (III):

i

(H)

where:

R ³ is a C ₁ -C ₁₀ alkyl or a group selected from (CH ₂ CH ₂ O) _n CH ₂ , where n has a value of 0 to 10, or (CH ₂ ) ₄ CH (NH ₂ ) COOCH ₂ ;

R ⁴ is selected from the group comprising O, C (O) R ¹ , R ¹ C (O) R ¹ or

R ¹ C (S) R ¹ (where R ¹ is O or NH); and m has the value from 1 to 11.

7. Compound according to claim 6, wherein m has the value of 1 to 3.

8. Compound according to any of claims 6 or 7, wherein R ³ is CH ₂ .

9. Compound according to any of claims 1 to 8, wherein the porous support is silica.

10. Compound according to any of claims 1 to 9, of formula (Vl):

(Vl)

11. Method of obtaining a compound described in any one of claims 1 to 10, comprising the following steps:

a) functionalization of the porous support using a compound comprising an azido (N3) or alkyne (C≡CH) terminal group.

b) functionalization of biotin or any of its derivatives with an alkyne (C≡CH) or azido (N3) terminal group;

c) reaction of the functionalized support of step (a) with the functionalized biotin or any of its functionalized derivatives of step (b) by reaction of 1, 3-dipolar cycloaddition ("click-chemistry").

12. Method according to claim 11, wherein the cycloadiction reaction occurs between an azido group of the functionalized porous support and an alkyne group of the functionalized biotin.

13. Use of a compound described in any of claims 1 to 10, for the purification of avidin.

14. Use of a compound described in any of claims 1 to 10, for the immobilization of avidin.

15. A complex comprising a compound described in any one of claims 1 to 10 and avidin.

16. Use of the complex described in claim 15, for the purification of biotinylated ligands.

17. Use of the complex described in claim 15, for fluorescent avidin marking.