阅读说明：本技术 锌铁纳米材料在降解突变p53蛋白中的应用 (Application of zinc-iron nano material in degradation of mutant p53 protein ) 是由 温龙平 张云娇 钱洁颖 张文彬 魏鹏飞 于 2020-06-04 设计创作，主要内容包括：本发明公开了一种锌铁纳米材料在降解突变p53蛋白中的应用。本发明首次通过工程化手段调控不同锌铁比例来有效对突变p53蛋白的降解,利用降解突变型p53联合化疗药物治疗及核磁共振成像实现p53耐药肿瘤的诊疗一体,为精准治疗及手术导航提供较好的借鉴,为纳米材料用于治疗突变p53肿瘤提供一种新的策略。本发明中还发现酶抑制剂、E1泛素活化酶、内吞抑制剂、TPEN、抗氧化剂、氧化酶抑制剂等能够该抑制锌铁纳米材料降解突变p53蛋白,且发现该锌铁纳米材料能够用于清除突变型p53蛋白外,还能有效地增加化疗药物顺铂的肿瘤杀伤效果,因此,可以将锌铁纳米材料作为化疗药物增敏剂,或与化疗药物联用用于抗肿瘤方面。(The invention discloses an application of a zinc-iron nano material in degradation of mutant p53 protein. The invention effectively degrades mutant p53 protein by regulating different zinc-iron ratios through an engineering means for the first time, realizes the diagnosis and treatment of p53 drug-resistant tumor by using the degradation mutant p53 in combination with chemotherapeutic drug therapy and nuclear magnetic resonance imaging, provides better reference for accurate therapy and surgical navigation, and provides a new strategy for the nano material to treat the mutant p53 tumor. The invention also finds that an enzyme inhibitor, E1 ubiquitin activating enzyme, an endocytosis inhibitor, TPEN, an antioxidant, an oxidase inhibitor and the like can inhibit the zinc-iron nano material from degrading the mutant p53 protein, and finds that the zinc-iron nano material can be used for removing the mutant p53 protein and effectively increasing the tumor killing effect of the chemotherapeutic drug cisplatin, so that the zinc-iron nano material can be used as a chemotherapeutic drug sensitizer or used in the anti-tumor aspect together with the chemotherapeutic drug.)

1. The application of the zinc-iron nano material in degrading the mutant p53 protein is characterized in that:

the environment of the application is in vitro environment;

the mass ratio of zinc element to iron element in the zinc-iron nano material is 1: 2-1: 18.

2. The use of zinc-iron nanomaterial according to claim 1 for degrading mutant p53 protein, wherein:

the mass ratio of zinc element to iron element in the zinc-iron nano material is 1: 2;

the zinc-iron nano material is spherical zinc-iron nano particles, and the average particle size of the zinc-iron nano particles is 10 nm;

the mutant p53 protein is at least one of p53 protein mutant S241F, E285K, Y220C, R249S, R280K, R248W, R175H, R273H and G245C.

3. The application of the zinc-iron nano material according to any one of claims 1-2 in degrading mutant p53 protein, wherein the zinc-iron nano material is characterized in that:

the zinc-iron nano material is prepared by the following method:

dissolving zinc oleate and iron oleate in a mixed solution of 1-octadecene and oleic acid, reacting at 120-320 ℃ in vacuum or protective gas atmosphere, cooling after the reaction is finished, adding isopropanol with the volume of 2-4 times of that of the mixture, centrifuging after the mixture is uniformly mixed, discarding the supernatant, washing the precipitate with isopropanol, and obtaining a zinc-iron nano material;

the mass ratio of the zinc element in the zinc oleate to the iron element in the iron oleate is 1: 2-1: 18;

the dosage of the oleic acid is 1/2 which accounts for the total molar weight of the zinc oleate and the iron oleate;

the reaction conditions are as follows: the reaction was carried out at 120 ℃ under vacuum or protective gas atmosphere for 30 minutes and then at 320 ℃ under vacuum or protective gas atmosphere for 2 hours.

4. The application of the zinc-iron nanomaterial of claim 3 in degrading mutant p53 protein, further comprising a step of transferring the obtained zinc-iron nanomaterial into a water phase, specifically:

ultrasonically dispersing the obtained zinc-iron nano material in n-hexane, adding sodium citrate, acetone and ultrapure water, carrying out water bath reaction at 70 ℃, adding acetone with the volume of 2-4 times of that of the mixture after the two-phase exchange reaction is finished, uniformly mixing, centrifuging, washing, and finally ultrasonically dispersing into water;

the reaction time is 3-5 hours.

5. The application of the zinc-iron nanomaterial of claim 3 in degrading mutant p53 protein, further comprising a step of transferring the obtained zinc-iron nanomaterial into a water phase, specifically:

the iron oleate is prepared by the following method: adding ferric trichloride and sodium oleate into a mixed solution of ethanol, normal hexane and ultrapure water, stirring and reacting in an oil bath kettle at 70 ℃, cooling after the reaction is finished, washing, collecting an upper-layer ferric oleate solution, and standing or performing rotary evaporation to volatilize the normal hexane to obtain ferric oleate;

the molar ratio of the ferric trichloride to the sodium oleate is 1: 3;

the volume ratio of the ethanol to the n-hexane to the ultrapure water is 4:6: 3;

the reaction time is 2-4 hours;

the washing is carried out by adopting ultrapure water;

the standing conditions are as follows: standing at room temperature overnight;

the zinc oleate is prepared by the following method: adding zinc chloride and sodium oleate into a mixed solution of ethanol, normal hexane and ultrapure water, stirring and reacting in an oil bath kettle at 70 ℃, cooling after the reaction is finished, washing, collecting an upper-layer zinc oleate solution, standing or performing rotary evaporation to volatilize normal hexane to obtain zinc oleate;

the molar ratio of the ferric trichloride to the sodium oleate is 1: 2;

the volume ratio of the ethanol to the n-hexane to the ultrapure water is 4:6: 3;

the reaction time is 2-4 hours;

the washing is carried out by adopting ultrapure water;

the standing conditions are as follows: the mixture was left at room temperature overnight.

6. Use of the zinc-iron nanomaterial described in any one of claims 1 to 5 in the preparation of a drug for degrading mutant p53 protein.

7. Use of at least one of an enzyme inhibitor, E1 ubiquitin activating enzyme, endocytosis inhibitor, N' -tetrakis (2-picolyl) ethylenediamine, antioxidant, oxidase inhibitor in the preparation of a product for inhibiting the degradation of mutant p53 protein by zinc-iron nanomaterial as claimed in any one of claims 1 to 5, wherein:

the enzyme inhibitor is a proteasome inhibitor MG-132;

the endocytosis inhibitor is an endocytosis inhibitor Genistein;

the antioxidant is antioxidant N-acetylcysteine;

the oxidase inhibitor is an oxidase inhibitor VAS 2870.

8. The use of the zinc-iron nanomaterial of any one of claims 1 to 5 in combination with a chemotherapeutic agent in the preparation of an anti-tumor agent, wherein:

the chemotherapy drug is cisplatin.

9. An antitumor agent characterized by: the zinc-iron nano material and the chemotherapeutic drug as defined in any one of claims 1 to 5, wherein: the chemotherapy drug is cisplatin.

10. The use of the zinc-iron nanomaterial described in any of claims 1 to 5 in the preparation of chemotherapeutic drug sensitizers or contrast agent materials for magnetic resonance imaging.

Technical Field

The invention belongs to the field of nano biomedicine, and particularly relates to an application of a zinc-iron nano material in degradation of mutant p53 protein.

Background

The p53 protein is a transcription factor encoded by the TP53 gene and regulates the transcription of a large number of target genes involved in cell cycle arrest, apoptosis and metabolism. TP53 has been mutated in more than 50% of human cancers, up to 96% in certain cancer subtypes such as high serous ovarian cancer, with the p53 mutation being the most common and most studied cancer suppressor gene. Most mutations of p53 are missense mutations, often occurring in the DNA binding region, leading to the expression and accumulation of highly stable mutant p53 protein. These mutants can be broadly divided into two categories: contact mutants and conformational mutants. In most cases, the mutation p53 lost the ability to interact with DNA binding sequences and therefore failed to activate the tumor suppressor function of p 53. In some cases, the mutation p53 may also act in other ways, thereby disabling the function of other tumor suppressors, such as p63 and p 73. In addition, an important characteristic of many mutant p53 is that new functions are obtained, and mutant p53 with the new functions obtained can promote the growth, metastasis, drug resistance and the like of tumors by regulating various key cellular processes from chromatin structure to metabolism.

Since tumor cells are largely dependent on the persistence of the mutation p53, the elimination of the mutation p53 is naturally a drug target for the treatment of p53 mutant cancers. Among the various strategies for cancer treatment directed to mutant p53, the most direct one is the clearance of mutant p53 protein by inducing degradation. Over the past two decades, much research has been devoted to the discovery of small molecule drugs to degrade mutant p53 proteins, primarily through the proteasome or autophagy pathways leading to degradation of mutant p53 proteins. In some reported examples, statins, NSC59984, Hsp90 inhibitors 17-AAG and ganetespib induced degradation of mutant p53 via the major proteasomal pathway, while MCB-613, SAHA, gamlogic acid and zn (ii) -curr degraded mutant p53 protein primarily via the autophagy pathway.

The research shows that wild type p53 needs to react with metal ion Zn ((S))²⁺) Can fold into the correct conformation. Mutant p53 protein lost the ability to bind zinc ions due to conformational changes. The conformational mutants G245C and G245D p53 are added with zinc in an exogenous manner, so that the wild type conformation can be partially restored, and the normal function of p53 in inducing cancer cell apoptosis is exerted. Thioimidazolidone metal ion chelate NSC31926 is used as a switch to chelate the zinc ion concentration in the culture environment to regulate the stability of mutant p53 protein. Curcumin zinc small molecule compounds cause conformational changes in R175H mutant p53 and induce degradation of mutant p53 proteins through the autophagosomal pathway by delivering zinc ions into cells. Therefore, the potential of zinc ions for restoring wild-type folded or degraded mutant p53 protein has been demonstrated, however, in addition to the above two small molecule compounds, exogenously added zinc ions are difficult to enter cells due to strict regulation of cell membrane ion channels and cause severe toxic reactions to cells when the concentration of zinc ions is more than 100. mu.M.

The zinc-iron nano material has a good T2 imaging function as an MRI contrast agent, but no reports about the use of the nano material in treating mutant p53 tumors exist so far.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the application of the zinc-iron nano material in degrading the mutant p53 protein.

The invention also aims to provide the application of the combination of the zinc-iron nano material and the chemotherapeutic drug in the preparation of the antitumor drug.

The purpose of the invention is realized by the following technical scheme: an application of a zinc-iron nano material in degrading mutant p53 protein.

The environment of the application is an in vitro environment.

The mass ratio of the zinc element to the iron element in the zinc-iron nano material is (1:2) - (1: 18); preferably 1:2.

The zinc-iron nano material is spherical zinc-iron nano particles, and the average particle size of the zinc-iron nano material is 10 nm.

The zinc-iron nano material is preferably prepared by the following method: dissolving zinc oleate and iron oleate in a mixed solution of 1-octadecene and oleic acid, reacting at 120-320 ℃ in vacuum or protective gas atmosphere, cooling after the reaction is finished, adding isopropanol with the volume of 2-4 times of that of the mixture, mixing uniformly, centrifuging, removing supernatant, washing the precipitate with isopropanol, and obtaining the zinc-iron nano material.

The mass ratio of the zinc element in the zinc oleate to the iron element in the iron oleate is (1:2) - (1: 18); preferably 1:2 (i.e. the molar ratio of zinc oleate to iron oleate is 0.11 to 1: 2.3; preferably 1: 2.3).

The amount of oleic acid is 1/2 based on the total molar amount of zinc oleate and iron oleate.

The reaction conditions are preferably: the reaction was carried out at 120 ℃ under vacuum or protective gas atmosphere for 30 minutes and then at 320 ℃ under vacuum or protective gas atmosphere for 2 hours.

The protective gas is preferably nitrogen.

The added isopropanol is preferably added in a volume of 3 times that of isopropanol.

The conditions of the centrifugation are preferably: centrifuge at 5000rpm for 10 minutes.

The number of washing times is more than 2.

The storage mode of the zinc-iron nano material is preferably as follows: the obtained zinc-iron nano material is dispersed in n-hexane by ultrasonic, and then stored in a refrigerator at 4 ℃.

The preparation method of the zinc-iron nano material further comprises the step of transferring the obtained zinc-iron nano material into a water phase, and specifically comprises the following steps:

ultrasonically dispersing the obtained zinc-iron nano material in n-hexane, adding sodium citrate, acetone and ultrapure water, carrying out water bath reaction at 70 ℃, adding acetone with the volume of 2-4 times of that of the mixture after the two-phase exchange reaction is finished, uniformly mixing, centrifuging, washing, and finally ultrasonically dispersing into water.

The acetone added is preferably added in a volume of 3 times that of the acetone.

The reaction time is 3-5 hours; preferably 4 hours.

The conditions of the centrifugation are preferably: centrifuge at 5000rpm for 10 minutes.

The number of washing times is more than 3.

The iron oleate is preferably prepared by the following method:

adding ferric trichloride and sodium oleate into a mixed solution of ethanol, normal hexane and ultrapure water, stirring and reacting in an oil bath kettle at 70 ℃, cooling after the reaction is finished, washing, collecting an upper-layer ferric oleate solution, and standing or performing rotary evaporation to volatilize the normal hexane to obtain the ferric oleate.

The molar ratio of the ferric trichloride to the sodium oleate is 1: 3.

The volume ratio of the ethanol to the n-hexane to the ultrapure water is 4:6: 3.

The stirring speed of the reaction is preferably 1000 rpm.

The reaction time is 2-4 hours; preferably 4 hours.

The washing is carried out by adopting ultrapure water.

The standing conditions are as follows: the mixture was left at room temperature overnight.

The zinc oleate is preferably prepared by the following method:

adding zinc chloride and sodium oleate into a mixed solution of ethanol, normal hexane and ultrapure water, stirring and reacting in an oil bath kettle at 70 ℃, cooling after the reaction is finished, washing, collecting an upper layer of zinc oleate liquid, standing or performing rotary evaporation to volatilize normal hexane, and obtaining zinc oleate.

The molar ratio of the ferric trichloride to the sodium oleate is 1:2.

The volume ratio of the ethanol to the n-hexane to the ultrapure water is 4:6: 3.

The stirring speed of the reaction is preferably 1000 rpm.

The reaction time is 2-4 hours; preferably 4 hours.

The washing is carried out by adopting ultrapure water.

The standing conditions are as follows: the mixture was left at room temperature overnight.

The mutant p53 protein is p53 protein coded by partial base mutation on p53 gene, and comprises a binding mutant p53 and a conformation mutant p 53; preferably at least one of p53 protein mutant S241F, E285K, Y220C, R249S, R280K, R248W, R175H, R273H and G245C; more preferably at least one of S241F, R249S, Y220C, R280K and R248W.

The effective concentration of the zinc-iron nano material is 20 mug/mL.

Application of zinc-iron nano material in preparation of drugs for degrading mutant p53 protein.

The application of at least one of an enzyme inhibitor, E1 ubiquitin activating enzyme, an endocytosis inhibitor, TPEN (N, N, N ', N' -tetra (2-picolyl) ethylenediamine), an antioxidant and an oxidase inhibitor in preparing a product for inhibiting zinc-iron nano material from degrading mutant p53 protein.

The product comprises a medicine, a kit and the like.

The enzyme inhibitor is preferably proteasome inhibitor MG-132.

The endocytosis inhibitor is preferably an endocytosis inhibitor Genistein.

The antioxidant is preferably the antioxidant NAC (N-acetylcysteine).

The oxidase inhibitor is preferably VAS 2870.

The zinc-iron nano material can be used as a mutant p53 protein degradation agent in the application of preparing antitumor drugs, and can inhibit the proliferation and migration capacity of cancer cells.

The zinc-iron nano material and the chemotherapeutic drug are combined to be applied to the preparation of the antitumor drug, and the synthesized zinc-iron nano material can be used for removing mutant p53 protein and effectively increasing the tumor killing effect of the chemotherapeutic drug cisplatin.

The chemotherapy drugs comprise cisplatin and the like.

The tumor comprises ovarian cancer, breast cancer and the like.

An anti-tumor drug comprises the zinc-iron nano material and a chemotherapeutic drug.

The tumor comprises ovarian cancer, breast cancer and the like.

The chemotherapy drugs comprise cisplatin and the like.

The application of the zinc-iron nano material in preparing chemosensitizer or contrast agent material for magnetic resonance imaging.

The chemotherapy drugs comprise cisplatin and the like.

Compared with the prior art, the invention has the following advantages and effects: the invention effectively degrades the mutant p53 protein by regulating different zinc-iron ratios through an engineering means for the first time, realizes the diagnosis and treatment of p53 drug-resistant tumor by using the mutant p53 in combination with chemotherapeutic drug therapy and nuclear magnetic resonance imaging, provides a better reference for accurate therapy and surgical navigation, and provides a new strategy for the nano material to treat the mutant p53 tumor.

Drawings

FIG. 1 is a graph of western blot verification results of zinc-iron nanomaterial-induced degradation mutant p53 with different Zn-Fe ratios obtained in example 1.

FIG. 2 is a graph showing the spectral analysis of the product obtained in example 1.

Figure 3 is the XRD pattern of the product obtained in example 1.

FIG. 4 is a photograph of a transmission electron microscope of the product obtained in example 1.

FIG. 5 is a graph of hydrated particle size of the product obtained in example 1.

FIG. 6 is a graph of the surface charge of the product obtained in example 1 in water.

FIG. 7 is an in vitro MRI image of the product obtained in example 1; wherein: a is a hysteresis loop diagram; b and C are T2 imaging signal values and imaging graphs of zinc-iron nano materials with different concentrations.

FIG. 8 is a graph of the in vivo MRI imaging effect of the product obtained in example 1.

FIG. 9 is a western blot result of ZnFe-4 nanomaterial induced degradation of mutant p53 protein in p53 cells containing different mutations.

FIG. 10 is a graph of western blot detection results of the ZnFe-4 nanomaterial on the degradation dosage and time dependence of the induced mutation p53 protein in different mutation p53 cells; wherein A is a graph of the result of the dose-dependent and time-dependent electrophoresis of ES-2 cells; b is a graph of the results of dose-and time-dependent electrophoresis of the BT549 cell line.

FIG. 11 is an immunofluorescence plot of the degradation of mutant p53 in ES-2 cells by ZnFe-4 nanomaterial.

FIG. 12 is a graph showing the result of western blot detection of p53 protein in wild-type p53 cells using ZnFe-4 nanomaterial; wherein A is a dose effect graph; b is a time effect graph.

FIG. 13 is a diagram showing the result of western blot detection of protein degradation of mutant p53 in H1299 cells with the ZnFe-4 nanomaterial out-transferred mutant p53(S241F) cells; wherein A is a dose effect graph; b is a time effect graph.

FIG. 14 is a statistical view of the fluorescent quantitative PCR results of ZnFe-4 nanomaterial on p53 gene of ES-2 cells.

FIG. 15 is a graph showing the results of p53 protein western blot of ZnFe-4 nanomaterial treated with CHX in ES-2 cells.

FIG. 16 is a graph showing the result of western blot of p53 protein after treatment of ZnFe-4 nanomaterial with 3-MA, MG-132 in ES-2 cells.

FIG. 17 is a graph showing the result of processing the ubiquitination level of ES-2 cells by ZnFe-4 nanomaterial; wherein A is a result graph of ubiquitination level in cells detected by western blot after 2h, 4h and 6h of ZnFe-4 nano material; b is a result graph of ubiquitination level on p53 protein detected by western blot after ZnFe-4 nano material and MG-132 are treated and p53 protein is immunoprecipitated.

FIG. 18 is a graph of p53 protein degradation in western blot detection cells after treatment of ZnFe-4 nanomaterial with PYR-41 in ES-2 cells.

FIG. 19 is a graph of p53 protein degradation in cells detected by western blot after ZnFe-4 nanomaterial is treated with endocytosis inhibitor Genistein in ES-2 cells.

FIG. 20 shows that after 2h, 4h and 6h of ES-2 cells are treated by ZnFe-4 nano-materials, a zinc ion probe FluoZin^TM-3 fluorescence map for detecting the intracellular zinc ion content.

FIG. 21 is a graph showing that degradation of p53 protein in cells is detected by western blot after ZnFe-4 nanomaterial is treated with metal ion chelating agent TPEN in ES-2 cells.

FIG. 22 is a graph showing the effect of ZnFe-4 nanomaterial on intracellular ROS levels; wherein A is a FACS detection and fluorescence map of total intracellular ROS; and B is a FACS detection result graph of mitochondrial ROS in the cell.

FIG. 23 is a graph showing the results of p53 degradation in cells detected by western blot after treatment of ZnFe-4 nanomaterial with ROS inhibitors NAC, Mito Tempo, VAS2870 in ES-2 cells.

FIG. 24 is a diagram showing the results of the CCK8 kit detecting the cell viability of each cell after different mutant p53 cell lines are treated by ZnFe-4 nano materials.

FIG. 25 is a graph showing the results of Annexin-V/PI FACS detection after ZnFe-4 nanomaterial treatment on different mutant p53 cell lines.

FIG. 26 is a graph showing the results of killing of p53 knocked-out ES-2 cells and ES-2 cells by ZnFe-4 nanomaterial; wherein A is a result graph of a western blot result of p53 gene knockout in ES-2 cells; b is a CCK8 result graph of ZnFe-4 nano-materials on p53 knocked-out ES-2 cells and ES-2 cells.

FIG. 27 is a fluorescent quantitative PCR image of p21 gene of ES-2 cells with ZnFe-4 nanomaterial.

FIG. 28 is a graph showing the results of p53 and p21western blot of ZnFe-4 nanomaterial on ES-2 cells.

FIG. 29 is a FACS result graph showing the effect of ZnFe-4 nanomaterial on the cell cycle of ES-2 cells.

FIG. 30 is a photograph of the effect of ZnFe-4 nanomaterial on ES-2 cell migration.

FIG. 31 is a photograph of the formation of ES-2 cell clones by ZnFe-4 nanomaterials.

FIG. 32 is a graph of killing of ZnFe-4 nanomaterials in ES-2 cells in combination with cisplatin treatment.

FIG. 33 is a graph showing the result of ICP detection of ZnFe-4 nanomaterial in animal body.

FIG. 34 is a graph showing the results of serum detection of mice injected with ZnFe-4 nanomaterial; wherein A is the concentration of AST (aspartate aminotransferase); b is the concentration of ALT (alanine aminotransferase); c is the concentration of BUN (blood urea nitrogen); d is the concentration of SCR (serum creatinine).

FIG. 35 is a graph showing the result of H & E staining of mouse organs after mice were injected with ZnFe-4 nanomaterial.

FIG. 36 is a graph of the statistical results of the body weight of mice treated with ZnFe-4 nanomaterial and ES-2 tumor bearing mice.

FIG. 37 is a graph showing the change of tumor volume after the ZnFe-4 nanomaterial is used for treating ES-2 tumor-bearing mice.

FIG. 38 is a photograph of tumors after the ZnFe-4 nanomaterial is used for treating ES-2 tumor-bearing mice.

FIG. 39 is a weight chart of tumors of mice bearing ES-2 tumor treated by ZnFe-4 nanomaterial.

FIG. 40 is a graph of the statistical results of the body weight of mice treated with ZnFe-4 nanomaterial after PDX tumor-bearing mice.

FIG. 41 is a graph of the change of tumor volume after the ZnFe-4 nanomaterial is used for treating PDX tumor-bearing mice.

FIG. 42 is a photograph of tumor after the ZnFe-4 nanomaterial is used to treat PDX tumor-bearing mice.

FIG. 43 is a weight graph of tumor after the ZnFe-4 nanomaterial is used to treat PDX tumor-bearing mice.

FIG. 44 is a TUNEL staining result of tumor tissue after treatment of ZnFe-4 nanomaterial ES-2 tumor-bearing mice.

FIG. 45 is a graph of TUNEL staining results of tumor tissues after treatment of ZnFe-4 nanomaterial PDX tumor-bearing mice.

FIG. 46 is a graph of immunohistochemical staining results of tumor tissues after treatment of ZnFe-4 nanomaterial PDX tumor-bearing mice.

FIG. 47 is a graph showing the result of p53 protein western blot of tumor tissues after treatment of a ZnFe-4 nanomaterial PDX tumor-bearing mouse.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.