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  • 专利说明
  • 权利要求
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    • 专利摘要:
    Functionalised proteins, method for obtaining them and application in infrared detection. The present invention describes a novel methodology of functionalization of proteins by incorporating a metal fragment of Renium with high yield, giving rise to coordinated iminopyridines possessing three carbonyl groups in position fac- offering bands coming from the tension of the CO bond in the infrared spectrum (IR) in an area where proteins do not show any signal, which allows a very interesting application from the point of view of detection of molecules of biological interest. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2713260A1
    申请号:ES201731332
    申请日:2017-11-16
    公开日:2019-05-20
    发明作者:Miguel Lucía Alvarez；San Juan Héctor Barbero；Rodríguez Raúl García；González Celedonio Manuel Alvarez；San José Daniel Miguel；Alvarez José Miguel Martín
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    [0001]
    [0002] FUNCTIONALIZED PROTEINS, METHOD FOR THEIR OBTAINATION AND APPLICATION IN INFRARED DETECTION
    [0003]
    [0004] The present invention falls within the field of bioinorganics and refers to a new method of protein functionalization through the incorporation of a metal fragment containing carbonyl groups capable of being observed by infrared spectroscopy (IR).
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007]
    [0008] Bioinorganic chemistry is a field of inorganic chemistry of growing scientific interest, especially in relation to the study of protein-metal fragment complexes in which there exists at least one covalent bond that unites the biomolecule and the prostate group. Said metal fragment can offer information about the protein (for example, its spatial location in a biological sample) by means of various spectroscopic techniques. In this context, Renio metal occupies an important place, most likely due to its possible use as a radiopharmaceutical or with applications for bioimaging. However, all the studies carried out to date use complexes fac-triacuo (or precursors thereof) and the metal load is obtained mainly by coordination of the imidazole group of the side chain of the amino acid histidine (VerZiegler, CJ, Herrick, RS; Rowlett, RS Chem. Commun.
    [0009] 2010 , 46, 1203-1205) The metal load obtained in the exposed cases is very low. Furthermore, if it is desired to use the metal fragment as an observable sensor by UV-Visible emission spectroscopy, a post-functionalization step is necessary to bind a chromophoric ligand to the metal.
    [0010]
    [0011] Another possibility resides in the functionalization of the protein through reactions in the ligands that are part of the first sphere of coordination of the metal that are activated by it. Thus, a good example is the Schiff reaction between a primary amine and a coordinated aldehyde, such as 2-pyridinecarboxaldehyde. There are studies in which it can be demonstrated that it is possible to join a biomolecule (peptide s) to a fac-tricarbonyl fragment of rhenium containing the ligand 2-pyridinecarboxaldehldo giving rise to the corresponding iminopyridines (see Alvarez, CM, Garcla-Rodrlguez, R .; Miguel, D. Inorg, Chem. 2012 , 51, 2984 2996).
    [0012]
    [0013] From the aforementioned work it can be concluded that the aldehyde group reacts exclusively with non-deactivated primary amines, never with secondary amines or amide groups. The only amino acid whose amino terminal group fulfills these characteristics, within the group of amino acids that are part of natural proteins, is that of Lysine.
    [0014]
    [0015] DESCRIPTION OF THE INVENTION
    [0016]
    [0017] The present invention describes two methods by which it is possible to load a protein (functionalize it) with a fragment of Renium with high yield, where the protein must possess amino acids Lysine in its structure. Both methods remarkably improve the metal loading capacity in a protein compared to the current state of the art. One is carried out under conditions that are soft enough so as not to denature the biomolecule, and another under more energetic conditions to achieve a maximum of practically complete metal loading.
    [0018]
    [0019] The selected Rhenium fragment is the metal / ac-tricarbonyl fragment of Renium, since the existence of the CO ligands provide clear and infra-red defining signals of the existence of the coordination complex in the protein. The aforementioned CO ligands offer very acute signals precisely in the area between 1800 cm-1 and 2500 cm-1 in an infrared absorption spectrum (IR), which is always valid in a protein. This constitutes a remarkable advance in the detection of functionalized infrared (IR) proteins due to the advantages over other techniques (cheap, fast, easy availability, seldom signal is lost due to structural changes in the environment of the coordination complex, etc.). Furthermore, in this way post-functionalization steps are avoided since the metal fragment incorporated into the protein already has the appropriate ligands acting as sensors.
    [0020]
    [0021] These two methods of functionalizing proteins with the complex [ReCl (CO) 3 (pyca)] are carried out through the Schiff reaction between the 2-pyridinecarboxaldehyde ligand and at least one amino group of the amino acids Lysine which is found in the corresponding protein, together with the terminal amino group thereof, provide iminopyridines of Formula I
    [0022]
    [0023]
    [0024]
    [0025]
    [0026] where P 'is the modified protein as a result of said reaction; and n is an integer number between 1 and the number of lysins contained in the starting protein.
    [0027]
    [0028] In an exemplary embodiment, these methods are carried out using the starting protein is bovine serum albumin (BSA). This protein has been selected because it is easily accessible since it belongs to the most abundant group of plasma proteins and comes from the waste of the livestock industry. It has in its structure 60 Lysins that, adding the terminal amino group of the protein, make a total of 61 groups susceptible to react with the complex [ReCl (CO) 3 (pyca)]. According to both preferred embodiments, said metal complex is combined with the protein in a proportion of 61 to 1, respectively.
    [0029]
    [0030] The result of the functionalization of this protein with the complex [ReCl (CO) 3 (pyca)] through the Schiff reaction between the 2-pyridinecarboxaldehyde ligand coordinated to the Renium and at least one amino group of the bovine serum albumin ( BSA) is a compound of formula II.
    [0031]
    [0032]
    [0033] where n is a value between 1 and 60, and BSA 'is the albumin protein of bovine serum modified after the Schiff reaction.
    [0034]
    [0035] It is interesting to highlight the applicability of this reaction for other proteins of interest from a biomedical point of view, such as phosphoinositide-3-kinase (PI3K), protein kinase B (PKB), rapamycin target in mammalian cells complex 1 (mTORCI). ), protein 9 of union to retinoblastoma (RBBP9), factor Inducible by Hypoxia-1 (HIF-1), acetylcholinesterase (ACHE), receptors of N-methyl-D-aspartate (NMDA), beta-secretase 1 (BACE1), and alpha-secretases, which participate in cancer-causing processes and in diseases such as Alzheimer's.
    [0036]
    [0037] In a first aspect, the present invention relates to the compound of formula I
    [0038]
    [0039]
    [0040]
    [0041]
    [0042] characterized in that P 'is a modified protein as a result of the reaction of Schiff between at least one amino group of the starting protein and the 2-pyridinecarboxaldehyde ligand (pyca) of the complex [ReCl (CO) 3 (pyca)], wherein the starting protein comprises at least one lysine; and n is an integer number between 1 and the number of lysines contained in the starting protein.
    [0043]
    [0044] In a preferred embodiment, the starting protein is selected from bovine serum albumin protein (BSA), phosphoinositide-3-kinase (PI3K), protein kinase B (PKB), rapamycin target in mammalian cells complex 1 (mTORCI). ), 9 binding to retinoblastoma protein (RBBP9), Hypoxia-1 inducible factor (HIF-1), acetylcholinesterase (ACHE), N-methyl-D-aspartate (NMDA) receptors, betasecretase 1 (BACE1), and alpha -secretasas.
    [0045]
    [0046] In a more preferred embodiment, the starting protein is bovine serum albumin (BSA).
    [0047]
    [0048] Another aspect of the present invention relates to the method of obtaining the compound of formula I, which comprises the Schiff reaction between at least one amino group of the starting protein and the 2-pyridinecarboxaldehyde ligand (pyca) of the complex [ReCl (CO. ) 3 (pyca)], wherein the starting protein comprises at least one lysine.
    [0049]
    [0050] In a preferred embodiment, the reaction occurs under mild conditions, so that the denaturing of the protein is prevented. Thus, both compounds are combined in the presence of tetrahydrofuran (THF) as a solvent in an inert atmosphere. The resulting mixture is stirred at room temperature protected from light for a time ranging from 1 to 1920 h. After the desired time has elapsed, the solvent is evaporated and the residue is washed with dichloromethane (DCM) to remove the complex [ReCl (CO) 3 (pyca)] that has not reacted.
    [0051]
    [0052] In another preferred embodiment, the reaction occurs under more energy conditions, but relevant from the physiological point of view. Both compounds are mixed in the presence of Normal Saline Serum (aqueous solution of 0.9% sodium chloride (w / v)). The mixture is placed in an ultrasonic bath and sonicated in order to disperse the metal complex particles to the maximum. Subsequently, the sample is subjected to microwave radiation (MW) at a temperature between 35 ° C-40 ° C stirring at a speed of 1200 rpm for a time that can range between 1 and 4 h. A yellow precipitate appears. To the mixture is added dichloromethane (DCM), both phases are stirred and the organic phase is discarded to remove the excess [ReCl (CO) 3 (pyca)] complex. The solid is then filtered, washed with water and dried in vacuo.
    [0053]
    [0054] A third aspect of the invention relates to a method for the detection of proteins comprising the obtaining of the compound of formula I as described above, and the subsequent measurement by infrared spectroscopy.
    [0055]
    [0056] A fourth aspect of the invention is the use of the complex [ReCl (CO) 3 (pyca)], for the detection of proteins comprising at least one lysine, by infrared spectroscopy.
    [0057]
    [0058] In a preferred embodiment, the protein is selected from bovine serum albumin protein (BSA), phosphoinositide-3-kinase (PI3K), protein kinase B (PKB), rapamycin target in mammalian cells complex 1 (mTORC1), protein 9 of union to retinoblastoma (RBBP9), factor Inducible by Hypoxia-1 (HIF-1), acetylcholinesterase (ACHE), receptors of N-methyl-D-aspartate (NMDA), betasecretase 1 (BACE1), and alpha-secretases .
    [0059]
    [0060] In a more preferred embodiment, the protein is bovine serum albumin (BSA).
    [0061]
    [0062] Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0063]
    [0064] DESCRIPTION OF THE FIGURES
    [0065]
    [0066] FIG. 1 IR spectrum in tetrahydrofuran (THF) of the washing waters from the first preferred preparation mode (above) and in dichloromethane (DCM) of the washing waters from the second preferred preparation mode (below). He observe the bands corresponding to the carbonyls groups of the starting [ReCl (CO) 3 (pyca)] complex thus ensuring an adequate elimination of the excess reagent.
    [0067]
    [0068] FIG. 2 IR spectra in the solid state of the BSA protein (top), the starting complex [ReCl (CO) 3 (pyca)] (center) and the loaded protein after carrying out the second preferred mode of preparation (below). The presence of the metal fragment bound to the protein by the appearance of the bands corresponding to the coordinated carbonyls in the area between 1800 and 2500 cm-1 is shown in contrast to the absence of signals in the spectrum of the pure protein. In addition, the frequency of the bands varies slightly due to the formation of iminopyridine.
    [0069]
    [0070] FIG. 3 Variation of the percentage of metallic charge deduced from the analyzes by ICP-MS as a function of the reaction time for the first preferred preparation mode (above) and for the second preferred preparation mode (below).
    [0071]
    [0072] EXAMPLES
    [0073]
    [0074] The invention will be illustrated below by means of tests carried out by the inventors, which highlights the effectiveness of the product of the invention.
    [0075]
    [0076] Example 1. Soft reaction conditions
    [0077]
    [0078] The complex [ReCl (CO) 3 (pyca)] (20 mg, 0.052 mmol) (prepared according to the publication Alvarez, CM, Garda-Rodriguez, R., Miguel, D. Dalton Trans. 2012) is mixed in a schlenk flask . , 51, 2984-2996), the BSA protein (56.9 mg, 8.56x10-4 mmol) and stirred with a magnetic bar. The mixture is deoxygenated and placed in an inert atmosphere of nitrogen gas. After 15 mL of THF are added. The whole is deoxygenated and placed in the inert atmosphere of N2 again. Finally, it is left stirring at room temperature protecting the flask from the light for a time that can range between 1 and 1920 h. Once the desired time has elapsed, the magnetic bar is removed and the solvent is evaporated at a vacuum. The resulting residue is washed with dichloromethane (3 x 10 mL) by removing the wash water with the aid of a cannula. In said solvent, the Renium complex is soluble and the BSA protein is not, as can be deduced from Figure 1. Lastly, the solid is dried in a vacuum to give a colored protein yellow whose intensity will depend on the amount of metal load it supports.
    [0079]
    [0080] The new functionalized protein is insoluble in conventional organic solvents and in water. Qualitatively the presence of the metal fragment can be observed by the infrared (IR) spectrum in which the bands of tension CO that correspond to a fragment / ac-tricarbonilo appear clearly, as shown in Figure 2. Quantitatively, the amount of Renium incorporated into the biomolecule by an inorganic elemental analysis provided by Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
    [0081]
    [0082] This reaction was carried out several times using the same initial conditions and analyzing the result at different times after the purification procedure described above, from 1 hour to 1920 (80 days). The maximum percentage reached was around 44%, corresponding to a functionalization of 26 Lysines, as shown in Figure 3. The rest of the results are listed in Table 1.
    [0083]
    [0084]
    [0085]
    [0086]
    [0087]
    [0088] Example 2. Reaction energetic conditions
    [0089]
    [0090] The complex [ReCl (CO) 3 (pyca)] (10 mg, 0.026 mmol), the BSA protein (28 mg, 4.3x10-4 mmol) and a magnetic bar are placed in a vial designed for a microwave reactor. shake. 5 mL of Normal Saline Serum is added, the resulting suspension is capped and sonicated in the ultrasound bath for 3 minutes. After this, the vial is placed in the microwave reactor and the mixture is irradiated at a temperature of 37 ° C with stirring at a speed of 1200 rpm for a time that can range from 1 to 4 h. Once the desired time has elapsed, the vial is extracted, which contains a precipitate inside it, is uncovered and the magnetic bar is removed. The content is Transfer to a round flask with one mouth and add 5 mL of dichloromethane. The biphasic mixture is stirred vigorously and, after allowing to stand, the organic phase is removed with the aid of a Pasteur pipette. This procedure is done twice more. Finally, the solid is filtered through the use of a cannula and washed with distilled water (3 x 5 mL) to finally dry it in vacuo.
    [0091]
    [0092] The resulting solid has the same characteristics as those observed by the method described in Example 1, and the incorporation of the complex to the protein can be analyzed by infrared (IR) spectrum (Figure 2) and thanks to ICP-MS, a load is reached maximum of around 92% (Figure 3), corresponding to 55 functionalized Lysins (of 60 in total). With this preferred mode of realization, it can be deduced that practically all the Lysine amino acids that make up the BSA protein have been derivatized, both those accessible on the external surface thereof and those that are not. This leads to the conclusion that there has probably been a change in the tertiary structure of the biomolecule (denaturation) that has allowed the coordination complex to access the more internal Lysines. The rest of the results are shown in Table 2.
    [0093]
    [0094]
    [0095]
    [0096]
    权利要求:
    Claims (13)
    [1]
    1. Compound of formula I

    [2]
    2. Compound according to claim 1, wherein the starting protein is selected from bovine serum albumin protein, phosphoinositide-3-kinase, protein kinase B, rapamycin target in mammalian cells complex 1, protein 9 binding to retinoblastoma , Factor Inducible by Hypoxia-1, acetylcholinesterase, receptors of N-methyl-D-aspartate, beta-secretase 1, and alpha-secretases.
    [3]
    3. Compound according to any of the preceding claims, wherein the starting protein is the albumin protein of bovine serum.
    [4]
    4. Compound according to claim 3, wherein n comprises integer values in the range of 1 to 60.
    [5]
    5. Method of obtaining the compound of formula I, comprising the Schiff reaction between at least one amino group of the starting protein and the ligand 2 pyridinecarboxaldehyde (pyca) of the complex [ReCl (CO) 3 (pyca)], wherein the starting protein comprises at least one lysine.
    [6]
    6. Method according to claim 5, wherein the Schiff reaction is carried out in tetrahydrofuran, at a temperature between 20-30 ° C, and for a time between 1 and 1920 hours.
    [7]
    7. Method according to claim 5, wherein the Schiff reaction is carried out in an aqueous medium, at a temperature between 35 ° C-40 ° C, and for a time between 1 and 4 hours.
    [8]
    8. Method according to claim 7, wherein the reaction is carried out by microwave irradiation.
    [9]
    9. Method according to any of claims 5 to 8, characterized in that it comprises a step of purification after the Schiff reaction by washing with a halogenated solvent and final drying of the resulting solid.
    [10]
    10. Method for the detection of proteins comprising the obtaining of the compound of formula I according to claims 1 to 4 and the subsequent measurement by infrared spectroscopy.
    [11]
    11. Use of the complex [ReCl (CO) 3 (pyca)], for the detection of proteins comprising at least one lysine, by infrared spectroscopy.
    [12]
    12. Use according to claim 11, wherein the protein is selected from bovine serum albumin protein, phosphoinositide-3-kinase, protein kinase B, rapamycin target in mammalian cells complex 1, protein 9 binding to retinoblastoma, factor Inducible by Hypoxia-1, acetylcholinesterase, N-methyl-D-aspartate receptors, beta-secretase 1, and alpha-secretases.
    [13]
    13. Use according to claims 11 or 12, wherein the protein is bovine serum albumin.
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    同族专利:
    公开号 | 公开日
    WO2019097101A1|2019-05-23|
    ES2713260B2|2020-10-02|
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    申请号 | 申请日 | 专利标题
    ES201731332A|ES2713260B2|2017-11-16|2017-11-16|FUNCTIONALIZED PROTEINS, METHOD FOR THEIR OBTAINMENT AND APPLICATION IN INFRARED DETECTION|ES201731332A| ES2713260B2|2017-11-16|2017-11-16|FUNCTIONALIZED PROTEINS, METHOD FOR THEIR OBTAINMENT AND APPLICATION IN INFRARED DETECTION|
    PCT/ES2018/070735| WO2019097101A1|2017-11-16|2018-11-14|PROTEINS FUNCTIONALISED WITH THE [ReCl3(ryca] COMPLEX, METHOD FOR OBTAINING SAME AND USE IN INFRARED DETECTION|
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