A method for ligand-promoted olefination of tyrosine compounds

Abstract

The application relates to the technical field of organic synthesis, and discloses a tyrosine compound olefination reaction method based on ligand promotion. The application successfully modifies tyrosine compounds by using compounds containing ethenyl as terminal groups by taking tyrosine as an embedded guiding group and by optimizing reaction raw materials and reaction conditions, and provides a method for C-H functionalization modification and reaction of polypeptide macromolecules. The reaction provided by the application can be carried out as long as the peptide molecule contains tyrosine, and tyrosine does not need to be used as a terminal residue of the peptide molecule, so that the olefination reaction method provided by the application has a wide application range.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and in particular to a tyrosine compound olefination reaction method based on ligand promotion. BACKGROUND

[0002] Peptides as an important bioactive molecule, in medicinal chemistry and chemical biology, especially in protein-protein interactions, are attracting more and more attention. However, polypeptide therapy is seriously hindered by inherent limitations such as low membrane permeability, short half-life and low bioavailability. Reasonable chemical modification of polypeptides usually enhances biological activity and pharmacokinetics.

[0003] In recent years, some methods based on post-modification of Trp (tryptophan) residues have been successfully developed. Transition metal-catalyzed C-H functionalization of complex molecules enables the direct conversion of ubiquitous C-H bonds in organic molecules into C-C bonds, which provides an effective method for post-functionalization and pegylation of peptides. However, the transition metal-catalyzed C-H functionalization of complex macromolecules has low site selectivity, requires external directing groups for reaction, and has low reaction yield.

[0004] Tyr residues are essential amino acids in many biochemical processes, drugs and natural compounds, so it is of great significance to modify polypeptides with Tyr residues as modification sites. However, in sharp contrast to tryptophan residues, there are few C-H functionalization based on tyrosine residues. Current Tyr C-H activation modification methods rely heavily on the introduction of auxiliary directing groups at the phenolic hydroxyl site, which increases the steps of introducing and removing directing groups, thereby reducing the overall efficiency of the modification strategy.

[0005] Currently, it is still a major challenge to directly modify the C-H activation of tyrosine residues in peptides without the help of additional directing groups. SUMMARY

[0006] In order to solve the technical problems of C-H functionalization modification and reaction of polypeptide macromolecules, the present application provides a tyrosine compound olefination reaction method based on ligand promotion. The present application successfully modifies tyrosine compounds into compounds with vinyl groups as terminal groups by using tyrosine as an internal directing group and optimizing reaction raw materials and reaction conditions. Therefore, a method for C-H functionalization modification and reaction of polypeptide macromolecules is provided.

[0007] The specific technical scheme of the present application is as follows:

[0008] In one aspect, the present application provides a ligand-promoted olefination method of a tyrosine compound. The olefination method is: adding the tyrosine compound and a compound with a vinyl group as a terminal group into a solvent to react, and adding a catalyst, a ligand and an oxidant into the solvent, wherein:

[0009] The tyrosine compound has the following structural formula:

[0010] R2 is selected from C1-C7 linear or branched alkoxy and C1-C7 linear or branched alkoxycarbonyl; and R3 is selected from C1-C7 linear or branched alkyl.

[0011] The compound with a vinyl group as a terminal group has the following structural formula:

[0012] R1 is selected from hydrogen, halogen, hydroxyl, amino, nitro, cyano, C1-C7 alkyl, C1-C7 alkoxy, C1-C7 alkylamino and C2-C7 unsaturated aliphatic hydrocarbon group.

[0013] The solvent is selected from AcOH, AcOH / THF mixed solvent, AcOH / DCE mixed solvent, AcOH / DMSO mixed solvent and AcOH / 1,4-dioxane mixed solvent.

[0014] The catalyst is selected from Pd(hfacac)2, PdCl2, Pd(acac)2, Pd(TMHD)2 and Pd(OPiv)2.

[0015] The oxidant is selected from tBuOOBz, Ag2O, H2O2, AgOAc, Cu(OAc)2, TBHP, BQ and O2.

[0016] The ligand is selected from 2-hydroxy-3,5-di(trifluoromethyl)pyridine and 2-hydroxy-3-nitro-5-trifluoromethylpyridine.

[0017] The present application successfully modifies the tyrosine compound by the compound with a vinyl group as a terminal group through the ligand by taking tyrosine as an embedded guiding group through preferred reaction raw materials and reaction conditions, and provides a method for C-H functional modification and reaction of a polypeptide macromolecule.

[0018] As a preferred reaction method, R2 is acetyl or tert-butoxycarbonyl, and R3 is methyl or ethyl.

[0019] As a preferred reaction method, the solvent is AcOH / 1,4-dioxane mixed solvent.

[0020] Further preferably, the volume ratio of AcOH to 1,4-dioxane in the AcOH / 1,4-dioxane mixed solvent is 1:1-10.

[0021] As a preferred reaction method, the catalyst is Pd(acac)2.

[0022] As a preferred reaction method, the oxidant is Cu(OAc)2.

[0023] As a preferred reaction method, the ligand is 2-OH-3-NO2-5-CF3 pyridine.

[0024] As a preferred reaction method, the reaction temperature is 80-120℃.

[0025] On the other hand, the present application provides a method for the directed modification of a polypeptide or protein, comprising the following steps:

[0026] Based on the above olefination reaction method of the tyrosine compound, a polypeptide or protein is chemically modified;

[0027] The polypeptide or protein contains tyrosine.

[0028] Compared with the prior art, the present application has the following technical effects:

[0029] (1) The present application successfully modifies the tyrosine compound into a compound with a vinyl group as the terminal group by using a ligand, by taking tyrosine as an internal guiding group and by optimizing the reaction raw materials and reaction conditions, thereby providing a method for the C-H functionalization modification and reaction of a polypeptide macromolecule.

[0030] (2) The tyrosine does not need to be the terminal residue of a peptide molecule, and the reaction provided by the present application can be performed as long as the peptide molecule contains tyrosine. The olefination reaction method provided by the present application has a wide application range. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The excitation spectrum and fluorescence emission spectrum of the reaction product 3a of the first group in Example 6 of the present application. DETAILED DESCRIPTION

[0032] The present application will be further described below in conjunction with examples. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the examples of the present application involved in the following descriptions are generally only examples of a part of the present application, rather than all examples. Therefore, all other examples obtained by those skilled in the art based on the examples in the present application without making creative efforts shall belong to the scope of protection of the present application.

[0033] The present application provides a ligand-promoted olefination method of tyrosine compound, comprising the following steps: step S1: adding tyrosine compound, compound with vinyl as terminal group, ligand, oxidant and catalyst into a pressure-resistant reaction bottle, at the same time, adding appropriate amount of reaction solvent, then sealing the reaction bottle and heating the reaction bottle to a certain temperature for reaction.

[0034] Step S2: after reaction, cooling the reaction bottle to room temperature, adding EtOAc to dilute the reaction solution, then sequentially washing the organic phase with pure water and saturated NaCl solution, then drying with anhydrous Na2SO4, collecting the liquid by decantation, concentrating by rotary evaporation to obtain solid, and finally purifying the product by flash column chromatography.

[0035] The reaction flow of the above reaction is as follows:

[0036]

[0037] Among them:

[0038] The structural formula of the tyrosine compound is:

[0039] R2 is selected from C1-C7 linear or branched alkoxy, C1-C7 linear or branched alkoxycarbonyl; R3 is selected from C1-C7 linear or branched alkyl;

[0040] The structural formula of the compound with vinyl as terminal group is:

[0041] R1 is selected from hydrogen, halogen, hydroxyl, amino, nitro, cyano, C1-C7 alkyl, C1-C7 alkoxy, C1-C7 alkylamino, C2-C7 unsaturated aliphatic hydrocarbon group;

[0042] The solvent is selected from AcOH, AcOH / THF mixed solvent, AcOH / DCE mixed solvent, AcOH / DMSO mixed solvent, AcOH / 1,4-dioxane mixed solvent;

[0043] The catalyst is selected from Pd(hfacac)2, PdCl2, Pd(acac)2, Pd(TMHD)2, Pd(OPiv)2;

[0044] The oxidant is selected from tBuOOBz, Ag2O, H2O2, AgOAc, Cu(OAc)2, TBHP, BQ, O2;

[0045] The ligand is selected from 2-hydroxy-3,5-di(trifluoromethyl)pyridine, 2-hydroxy-3-nitro-5-trifluoromethylpyridine;

[0046] The heating temperature is 80-120℃.

[0047] The above reaction method of the present application is further illustrated by the following specific examples.

[0048] Example 1

[0049] This example provides a ligand-promoted tyrosine compound olefination reaction method, and the reaction flow is as follows:

[0050]

[0051] Step S1: Put tyrosine compound 1a (0.1 mol), compound 2a with vinyl as the terminal group (0.3 mol), 2-hydroxy-3-nitro-5-trifluoromethylpyridine (3.1 mg, 0.015 mmol), Cu(OAc)2 (27.2 mg, 0.15 mmol) and Pd(acac)2 (3.05 mg, 0.01 mmol) into a pressure-resistant reaction bottle, at the same time, add 0.5 mL of AcOH / 1,4-dioxane (volume ratio 1:6) solution as the reaction solvent, then seal the reaction bottle, and heat the reaction bottle to 100℃ for reaction. After 10 hours of reaction, the reaction is completed.

[0052] Step S2: Cool the reaction bottle to room temperature, add 10 mL of EtOAc to dilute the reaction solution, then wash the organic phase with 15 mL of pure water and 15 mL of saturated NaCl solution in turn, then dry with anhydrous Na2SO4, collect the liquid by decanting, concentrate by rotary evaporation to obtain a solid, and finally purify the product 3a by flash column chromatography, with a yield of 78.9%.

[0053] According to nuclear magnetic analysis, the nuclear magnetic data of compound 3a is as follows:

[0054] 1H NMR (600 MHz, Chloroform-d) δ 7.94 (d, J = 16.0 Hz, 1H), 7.66 (s, 1H), 7.19 (s, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.57 (d, J = 16.1 Hz, 1H), 5.47 (d, J = 8.4 Hz, 1H), 5.09 (d, J = 8.4 Hz, 1H), 4.69 - 4.63 (m, 1H), 4.60 (d, J = 11.7 Hz, 1H), 4.54 (d, J = 7.9 Hz, 1H), 4.47 (d, J = 10.8 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.05 (dd, J = 14.2, 5.6 Hz, 1H), 2.94 (dd, J = 16.3, 6.6 Hz, 1H), 1.46 (s, 9H), 1.43 (s, 9H).

[0055] 13 C NMR (151 MHz, CDC13) δ 172.41, 170.51, 167.33, 155.36, 155.27, 141.71, 132.40, 130.09, 128.93, 127.70, 121.27, 117.16, 116.65, 80.49, 80.28, 64.30, 54.59, 53.12, 52.79, 52.33, 37.62, 28.29.

[0056] The nuclear magnetic resonance data of compound 3a are as follows by mass spectrometry analysis:

[0057] HRMS (ESI) m / z calcd for C 27 H 38 N2O 11 Na (M + Na) + 589.2368, found 589.2369.

[0058] Example 2

[0059] In this example, 15 groups of olefination reactions as shown in Example 1 were carried out with different reaction solvents (single variable).

[0060] The main difference between this example and Example 1 is that the 0.5 mL reaction solvents used in the 15 groups of olefination reactions of this example are as shown in Table 1. The others are the same as Example 1.

[0061] The yields of product 3a of the 15 groups of olefination reactions of this example are as shown in Table 1, respectively.

[0062] Table 1

[0063] Group Solvent 3a yield (%) 1 1,4-dioxane 0 2 DMF 0 3 THF 0 4 AcOH 20.3 5 AcOH / THF = 1:3 12.4 6 AcOH / H20 = 1 :3 0 7 AcOH / toluene = 1:3 0 8 AcOH / para-xylene = 1:3 0 9 AcOH / DCE = 1:3 15.2 10 AcOH / MeCN = 1:3 0 11 AcOH / DMSO = 1:3 16.0 12 AcOH / 1,4-dioxane = 1:3 73.2 13 AcOH / 1,4-dioxane = 1:1 71.4 14 AcOH / 1,4-dioxane = 1:6 78.9 15 AcOH / 1,4-dioxane = 1:10 72.1

[0064] From this example, when the reaction solvent is 1,4-dioxane, DMF, THF, no obvious product is observed, when AcOH is used as the solvent, 20.3% of the corresponding product is obtained, which shows that AcOH can promote the progress of the tyrosine ortho C-H activation reaction. AcOH / THF, AcOH / toluene, AcOH / para-xylene, AcOH / DCE, AcOH / MeCN and other 1:3 (v / v) mixed solvents do not improve the reaction yield, no target product is generated or very little is generated. But when AcOH is mixed with 1,4-dioxane, the reaction yield is increased to 73.2%, so it is guessed that AcOH and 1,4-dioxane may affect the reaction process of 1a and 2a in some way when they coexist. Further, this example confirms by experiment that the volume ratio of AcOH and 1,4-dioxane is preferably 1:6 (yield 78.9%).

[0065] Example 3

[0066] This example changes different ligands (single variable) to carry out 10 groups of olefination reactions as shown in Example 1.

[0067] The main difference between this example and Example 1 is that the 0.015 mmol ligands used in the 10 groups of olefination reactions in this example are as shown in Table 2. The others are the same as in Example 1.

[0068] The yields of the products 3a of the 10 groups of olefination reactions in this example are shown in Table 2, respectively.

[0069] Table 2

[0070]

[0071] From this example, when the ligand is ligand 3, ligand 4, the yield of compound 3a is low; when the ligand is ligand 5, ligand 10, the corresponding product is obtained with a yield of 66.0%, 78.9%. This example confirms by experiment that the most preferred ligand is 2-hydroxy-3-nitro-5-trifluoromethylpyridine (ligand 10).

[0072] Example 4

[0073] This example changes different oxidizing agents (single variable) to carry out 12 groups of olefination reactions as shown in Example 1.

[0074] The main difference between this example and Example 1 is that the 0.15 mmol oxidizing agents used in the 12 groups of olefination reactions in this example are as shown in Table 3. The others are the same as in Example 1.

[0075] The yield of product 3a of the 10 groups of olefination reactions of this example is shown in Table 3, respectively.

[0076] Table 3

[0077] Group Oxidizing agent 3a yield (%) 1 tBuOOBz 49.1 2 Ag2O 48.7 3 H2O2 42.3 4 AgOAc 45.6 5 [Cu(OAc)2] 78.9 6 TBHP 39.5 7 BQ 21.4 8 K2S2O8 0 9 AgF 0 10 CuBr 0 11 CuI 0 12 O2 43.6

[0078] It can be known from this example that when the oxidant is K2S2O8, AgF, CuBr, CuI, no obvious product is observed, and when Cu(OAc)2 is used as the oxidant, the yield reaches 78.9%.

[0079] Example 5

[0080] In this example, different catalysts (single variable) are used to perform 10 groups of olefination reactions as shown in Example 1.

[0081] The main difference between this example and Example 1 is that the 0.01 mmol catalysts used in the 10 groups of olefination reactions of this example are shown in Table 4, respectively. The others are the same as Example 1.

[0082] The yield of product 3a of the 10 groups of olefination reactions of this example is shown in Table 4, respectively.

[0083] Table 2

[0084] Group Catalyst 3a yield (%) 1 [Pd(OAc)2] 32.2 2 [Pd(hfacac)2] 65.3 3 [PdCl2] 63.0 4 [Pd(NBD)Cl2] 43.2 5 [Pd(acac)2] 78.9 6 [[Pd(MeCN)4](BF4)2] 21.4 7 [Pd(TMHD)2] 68.4 8 [PdBr2] 18.5 9 [Pd(OPiv)2] 62.2 10 [Pd(TFA)2] 31.0

[0085] It can be known from this example that when the catalyst is PdBr2, [Pd(MeCN)4](BF4)2, the yield of compound 3a is low; when Pd(acac)2 is used as the catalyst, the yield reaches 78.9%.

[0086] Example 6

[0087] In this example, different compounds 1a and different compounds 2a are used as raw materials to perform 10 groups of olefination reactions as shown in Example 1.

[0088] The main difference between this example and Example 1 is that the 10 groups of olefination reactions of this example use compounds 1a and compounds 2a as shown in Table 5, respectively. The others are the same as Example 1.

[0089] The yield of product 3a of the 10 groups of olefination reactions of this example is shown in Table 5, respectively.

[0090] Table 5

[0091]

[0092]

[0093] From this example, it can be seen that different sequences of dipeptides modified by acrylate (compound 2a) can also be successfully reacted with compound 1, and the corresponding products (compound 3a) are obtained in good yield. Different lengths and sequences of amino acids / peptides modified by vinyl benzamide (compound 2a) can also be successfully reacted with compound 1, and the corresponding products are obtained in moderate to good yield (reaction groups 5-6). As the product of reaction group 5, it has an aryl-alkene-aryl coupling structure and exhibits strong fluorescence in the range of 400-500 nm, with a maximum emission wavelength of 470 nm. Thus, one application of the olefination reaction method provided by the present application can be given.

[0094] The product 3a of reaction group 1, whose nuclear magnetic data are detailed in Example 1, can prove that its double bond is in the E-configuration. In addition, by using a light source of 345 nm to excite the olefination product 3a of reaction group 1, it is found that it exhibits strong fluorescence in the range of 400-600 nm, with a maximum emission wavelength of 470 nm, as shown in Figure 1 The excitation spectrum (left) and fluorescence emission spectrum (right) of compound 1a, compound 2a, and product 3a in reaction group 1. This means that the method can be used to conveniently construct fluorescent polypeptides, so as to visualize the role of bioactive peptides in signal transduction, metabolic regulation, and antibacterial and antiviral physiological processes in vivo. By modifying tyrosine through C-H olefination, polypeptides with built-in fluorescence properties can be synthesized, which can avoid the additional step of introducing fluorescent dyes and reduce the changes to the structure and function of polypeptides, especially cyclic peptides, so as to better study the functions of such tyrosine-containing polypeptides.

[0095] As can be seen from reactions 7-10, acrylate derivatives with natural product and drug skeletons can also be applied to C-H olefination reactions, thereby producing unique peptide / natural product and peptide / drug conjugates, which have good application value.

[0096] Example 7

[0097] The main difference between this example and Example 1 is that the reaction temperature of this example is 80°C. The others are the same as Example 1.

[0098] The yield of the olefination reaction product 3a of this example is 52%.

[0099] Example 8

[0100] The main difference between this example and Example 1 is that the reaction temperature of this example is 80°C. The others are the same as Example 1.

[0101] The yield of the olefination reaction product 3a of this example is 70.1%.

[0102] Comparative Example 1

[0103] The main difference between this comparative example and Example 1 is that Pd(acac)2 is not added. Other conditions are the same as Example 1.

[0104] No compound 3a is generated in this comparative example.

[0105] Comparative Example 2

[0106] The main difference between this comparative example and Example 1 is that the ligand 2-hydroxy-3-nitro-5-trifluoromethylpyridine is not added. Other conditions are the same as Example 1.

[0107] The yield of product 3a in the olefination reaction of this comparative example is 16.0%.

[0108] Comparative Example 3

[0109] The main difference between this comparative example and Example 1 is that Cu(OAc)2 is not added. Other conditions are the same as Example 1.

[0110] No obvious compound 3a is generated in this comparative example.

[0111] From the comparison of Comparative Examples 1-3 and Example 1, it can be seen that, in the present application, tyrosine is used as an internal guiding group, and a compound containing a vinyl group as a terminal group is modified to the ortho C-H of tyrosine in a polypeptide macromolecule containing tyrosine. The reaction conditions need to be controlled under certain conditions, so as to successfully modify and further obtain a higher yield. Further analysis shows that the olefination reaction provided in the present application should be carried out in a solvent, and the reaction solvent should be selected from AcOH, AcOH / THF mixed solvent, AcOH / DCE mixed solvent, AcOH / DMSO mixed solvent, and AcOH / 1,4-dioxane mixed solvent. In addition, a catalyst should be added to the olefination reaction provided in the present application, and the catalyst is selected from Pd(hfacac)2, PdCl2, Pd(acac)2, Pd(TMHD)2, and Pd(OPiv)2. An oxidizing agent is also added to the reaction, and the oxidizing agent is selected from tBuOOBz, Ag2O, H2O2, AgOAc, Cu(OAc)2, TBHP, BQ, and O2. At the same time, a ligand should be added to the reaction, and the ligand is selected from 2-hydroxy-3,5-di(trifluoromethyl)pyridine and 2-hydroxy-3-nitro-5-trifluoromethylpyridine. When the above conditions are met, the yield of the target product in the olefination reaction provided in the present application is higher.

[0112] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified. The methods used in the present application are conventional methods in the art unless otherwise specified.

[0113] The above is only the preferred embodiment of the present application, and does not limit the present application, and any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still belongs to the protection scope of the technical scheme of the present application.

Claims

1. A method for ligand-promoted olefination of tyrosine compounds, characterized by: The method comprises the following steps: Step S1: 0.1 mol of tyrosine compound 1a, 0.3 mol of compound 2a with a vinyl group as a terminal group, 0.015 mmol of 2-hydroxy-3-nitro-5-trifluoromethylpyridine, 0.15 mmol of Cu(OAc)2 and 0.01 mmol of Pd(acac)2 are added to a pressure-resistant reaction bottle, at the same time, 0.5 mL of an AcOH / 1,4-dioxane solution is added as a reaction solvent, then the reaction bottle is sealed and heated to 100 DEG C for reaction; wherein the volume ratio of AcOH to 1,4-dioxane in the AcOH / 1,4-dioxane solution is 1:6; Step S2: after 10 hours of reaction, the reaction bottle is cooled to room temperature, 10 mL of EtOAc is added to dilute the reaction solution, then the organic phase is washed with 15 mL of pure water and 15 mL of saturated NaCl solution in sequence, then dried with anhydrous Na2SO4, the liquid is collected by decanting, concentrated by rotary evaporation, a solid is obtained, and finally the product 3a is obtained by purification through flash column chromatography; Wherein: The 1a is selected from any one of the following compounds 1a, the 2a is selected from a compound 2a corresponding to the 1a, and the 1a and the corresponding compound 2a form a corresponding compound 3a:

2. A method of directed modification of a polypeptide or protein, characterized by: The method comprises the following steps: The method is used for chemical modification of a polypeptide or a protein according to the olefination reaction method of claim 1. The polypeptide or the protein contains tyrosine.

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