Tyrosine enol etherification reaction and its directed modification of tyrosine in polypeptides or proteins

Abstract

The present invention relates to the technical field of organic synthesis, and discloses a tyrosine enol etherification reaction and its directed modification of tyrosine in polypeptides or proteins. In the enol etherification reaction provided by the present invention, by reacting a tyrosine-containing compound with an olefin compound under certain conditions, the olefin compound can locate the phenolic hydroxyl group of the tyrosine-containing compound, react at a specific site, displace the hydrogen on the phenolic hydroxyl group, and obtain an enol etherified tyrosine compound. The method for the enol etherification reaction of the tyrosine-containing compound provided by the present invention does not require the introduction of a directing group, has simple operation, mild reaction conditions, and high site selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and particularly to the tyrosine enol etherification reaction and its directed modification of tyrosine in polypeptides or proteins. Background Art

[0002] Chemical modification refers to the installation of functional groups with specific functions onto target molecules through chemical reactions. Polypeptides or proteins are the main targets of chemical modification. Nature achieves the diversification of protein functions through a series of post-translational modifications, and these modifications in turn mediate the activity of proteins. The complex modifications in vivo limit the use of pure natural modified proteins, and the chemical modification of proteins has emerged as a result. Since chemical modification can improve the functions of polypeptides and proteins and endow them with new functions, the diversification of protein functions can also be achieved by introducing various different functional groups through chemical methods. The chemical modification of polypeptides or proteins has played an important role in life sciences, medicine, and biomaterials. In medicinal chemistry, drugs can improve their biological activity and bioavailability after chemical modification.

[0003] A polypeptide is a compound formed by 10 - 100 amino acids through dehydration condensation and linked together by peptide bonds. In drug research, natural sequence polypeptides are ideal choices. They are usually effective and selective agonists or antagonists of different receptors involved in pathological research. At the same time, compared with small molecule drugs, polypeptide drugs have higher receptor affinity and specificity and generally exhibit lower toxicity. However, polypeptide drugs also have some unfavorable characteristics such as poor absorption, delivery, metabolism, and excretion (ADME), and these unfavorable factors can be effectively improved by chemically modifying the polypeptide-containing drugs.

[0004] In the prior art, for example, Chinese Patent with the publication number CN115108953A discloses the thiocyanation reaction of alkynylamides and their selective modification of cysteine in polypeptides. In the polypeptide selective modification method provided by this patent, the reaction selectivity of the carbon-carbon triple bond in alkynylamides to the thiol group in cysteine is utilized to achieve the selective modification of cysteine in polypeptides and proteins. However, the modified product obtained by this method is a product with a specific cis single configuration.

[0005] Tyrosine is a non-essential amino acid with the chemical name 2-amino-3-(4-hydroxyphenyl)propanoic acid. It appears in natural proteins at a medium to low frequency and is an important amino acid involved in cell signaling. Many polypeptide drugs also contain tyrosine. Tyrosine has an electron-rich and hydrophobic phenol side chain, which is a good site for highly diverse biological modifications. In medicinal chemistry, the methods for modifying the tyrosine phenol side chain usually involve: first protecting the tyrosine phenolic hydroxyl group, then modifying other sites on the tyrosine phenolic hydroxyl group, and finally deprotecting to achieve the modification. These methods have relatively harsh conditions, many steps, and low reaction yields. In the modification of tyrosine-containing polypeptides, the methods targeting tyrosine modification usually involve esterification modification of the ortho-position of the tyrosine phenolic hydroxyl group, but there are problems such as instability of the ester bond and easy hydrolysis by esterase.

[0006] Therefore, developing a simple operation method for tyrosine chemical modification is of great significance in the field of protein modification. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a method for the enol etherification reaction of tyrosine-containing compounds and a method for the directed modification of polypeptides or proteins. The present invention creatively discovers that under certain reaction conditions, an olefin compound can locate the phenolic hydroxyl group of a tyrosine-containing compound, react at a fixed point, displace the hydrogen on the phenolic hydroxyl group, and obtain an enol etherified tyrosine compound with high site selectivity. At the same time, the present invention adjusts the reaction conditions so that the reaction has the characteristics of high efficiency under mild reaction conditions.

[0008] The specific technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a method for the enol etherification reaction of tyrosine-containing compounds, comprising the following steps:

[0010] A tyrosine-containing compound having the general structural formula (Ⅱ) reacts with an olefin compound having the general structural formula (Ⅲ) in a solvent under the action of a catalyst, an oxidant, an additive, and a base to obtain an enol etherified tyrosine compound having the general structural formula (Ⅰ):

[0011]

[0012] Wherein, R1 is selected from a straight-chain or branched-chain alkoxy group having 1 to 7 carbon atoms or a straight-chain or branched-chain alkoxycarbonyl group having 1 to 7 carbon atoms; R2 is selected from a straight-chain or branched-chain alkyl group having 1 to 7 carbon atoms; R3 is selected from a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a phenoxycarbonyl group, a chlorobenzene group, or a tert-butyl ether group.

[0013] The present invention creatively discovers that under certain reaction conditions, an olefin compound can target the phenolic hydroxyl group of a tyrosine-containing compound, undergo a reaction at a specific site, displace the hydrogen on the phenolic hydroxyl group, and obtain an olefinated tyrosine compound with high site selectivity. By reacting a tyrosine-containing compound with the structural formula (II) and an olefin compound with the structural formula (III) under certain conditions, the present invention can obtain an olefinated tyrosine compound with the structural formula (I). At the same time, by adjusting the reaction conditions, the reaction of the present invention has the characteristics of high efficiency under mild reaction conditions. The method for the olefination reaction of the tyrosine-containing compound provided by the present invention does not require the introduction of a directing group, has simple operation, mild reaction conditions, and high site selectivity.

[0014] As a preference of the above technical solution of the present invention, R1 is an acetyl group or a tert-butoxycarbonyl group, and R2 is a methyl group or an ethyl group; R3 is selected from a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a phenoxycarbonyl group, a p-methylphenoxycarbonyl group, a chlorobenzene group, or a tert-butyl ether group.

[0015] As a preference of the above technical solution of the present invention, the molar ratio of the tyrosine-containing compound, the olefin compound, the catalyst, the oxidant, the additive, and the base is 1:1 to 3:0.1 to 0.3:1 to 3:1 to 2:0.5 to 1.2.

[0016] The dosage ratio of the reaction substrates in a chemical reaction plays an important role in the progress of the chemical reaction. In the method of the present invention, a reasonable dosage ratio of the substrates in the olefination reaction can control the selectivity of the product, enable the olefin compound to react with the phenolic hydroxyl group of the tyrosine-containing compound at a specific site, preferentially generate the target product, and an appropriate dosage ratio of the substrates can make the reaction have a high reaction rate. In the method of the present invention, the preferred dosage of the substrates in the olefination reaction is 1:1 to 3:0.1 to 0.3:1 to 3:1 to 2:0.5 to 1.2. A dosage outside this range will result in a low reaction yield or the reaction cannot proceed.

[0017] As a preference of the above technical solution of the present invention, the catalyst is palladium acetate and / or palladium trifluoroacetate.

[0018] In the olefination reaction of the present invention, the catalyst is preferably selected from palladium acetate and palladium trifluoroacetate. The catalyst is the guarantee for the occurrence of the olefination reaction of the present invention. Under the catalysis of palladium acetate or palladium trifluoroacetate, the olefination reaction of the present invention has a good reaction effect, high reaction rate and yield. When the reaction occurs under the catalysis of other catalysts, such as tetrakis(triphenylphosphine)palladium, dichlorotriphenylphosphine palladium, and DPPF dichloropalladium, the reaction yield is relatively low.

[0019] As a preference of the above technical solution of the present invention, the oxidant is selected from one or more of potassium persulfate, silver acetate, and tert-butyl peroxybenzoate.

[0020] In the ene-etherification reaction of the present invention, the oxidant is preferably one or more selected from potassium persulfate, silver acetate, and tert-butyl peroxybenzoate. The role of the oxidant in the ene-etherification reaction of the present invention is to oxidize the catalyst to ensure the cyclic catalysis of the catalyst. In the presence of the oxidants potassium persulfate, silver acetate, or tert-butyl peroxybenzoate, the present ene-etherification reaction has a good reaction effect, with high reaction rate and yield. When adding other oxidants, such as oxygen, hydrogen peroxide, tert-butyl hydroperoxide, copper acetate, and silver acetate, the reaction yield is low. Among them, when adding copper acetate or silver acetate as the oxidant, the reaction yield is extremely low.

[0021] As a preference of the above technical solution of the present invention, the additive is preferably one or more selected from tetrabutylammonium bromide, tetrabutylammonium iodide, and tetrabutylammonium chloride.

[0022] In the ene-etherification reaction of the present invention, the additive is preferably one or more selected from tetrabutylammonium bromide, tetrabutylammonium iodide, and tetrabutylammonium chloride. The role of the above additive in the ene-etherification reaction of the present invention is to coordinate temporarily with the substrate to prevent the catalyst from being deactivated by coordination.

[0023] As a preference of the above technical solution of the present invention, the base is preferably one or more selected from potassium carbonate, cesium carbonate, potassium fluoride, and cesium fluoride.

[0024] In the ene-etherification reaction of the present invention, the base is preferably one or more selected from potassium carbonate, cesium carbonate, potassium fluoride, and cesium fluoride. The role of the base substance in the ene-etherification reaction of the present invention is to ionize the phenolic hydroxyl group on the tyrosine-containing compound and improve the reaction rate.

[0025] The present invention provides a method for the ene-etherification reaction of a tyrosine-containing compound. Its principle is as follows: Under the conditions of an additive and a base, the phenolic hydroxyl group of the tyrosine side chain in the tyrosine-containing compound is converted into a negative ion to obtain an intermediate compound A. Subsequently, palladium is oxidized and inserted to coordinate with the phenolic hydroxyl group to form an intermediate B. Then, the olefin compound adds and inserts with palladium to form a palladium-olefin complex. Subsequently, an addition migration occurs to form a new complex intermediate C. Finally, reductive elimination gives the product ene-etherified tyrosine compound.

[0026] As a preference of the above technical solution of the present invention, the solvent is dichloromethane.

[0027] As a preference of the above technical solution of the present invention, the reaction temperature is 20 - 55 °C, and the reaction time is 6 - 24 h.

[0028] In the ene-etherification reaction of the present invention, the reaction temperature is preferably 20 - 55 °C, and at this reaction temperature, the reaction has a good effect. The reaction time is preferably 6 - 24 h. Under this reaction time, the reaction can have a good reaction degree and a high yield.

[0029] As a preference of the above technical solution of the present invention, purification is carried out after the reaction. The purification method includes the following steps:

[0030] (1) Take the reaction product for diatomite filtration, then wash with DCM, then collect the organic phase, concentrate, extract with ethyl acetate and water, take the organic layer, dry, filter, and concentrate to obtain the crude product;

[0031] (2) Take the crude product for silica gel column chromatography, use a mixed solution of ethyl acetate and petroleum ether as the mobile phase, track by TLC and collect the eluate with an Rf value of 0.3 - 0.5. After removing the solvent from the eluate, an enol etherified tyrosine compound is obtained.

[0032] Furthermore, the volume ratio of ethyl acetate to petroleum ether is preferably 1:8 - 10.

[0033] On the other hand, the present invention also provides a method for site-directed modification of a polypeptide or protein, including the following steps: Based on the above-mentioned enol etherification reaction method, chemically modify the polypeptide or protein; wherein, the polypeptide or protein contains tyrosine.

[0034] In the enol etherification reaction methods disclosed in the prior art, it is usually an alkyne addition reaction, which has harsh reaction conditions, complex operations, and high difficulty in controlling the reaction sites, so the yield is low; at the same time, in the reported enol etherification reaction methods based on alkyne addition reactions, they are all carried out on small molecule compounds, and it is difficult to carry out the reaction on macromolecular substances or the yield of the enol etherified product is extremely low.

[0035] Based on the enol etherification reaction of tyrosine-containing compounds and olefin compounds, the present invention provides a method for site-directed modification of polypeptides or proteins, which can carry out site-directed enol etherification on polypeptides or proteins containing tyrosine, can carry out site-directed chemical modification on polypeptides or proteins, can be applied in medicinal chemistry to carry out site-directed chemical modification on polypeptide or protein drugs containing tyrosine, improve the biological activity of the drugs, and increase their bioavailability; it can also be applied in the labeling of polypeptides or proteins to achieve the tracing of polypeptides or proteins.

[0036] Compared with the prior art, the present invention has the following technical effects:

[0037] The present invention provides an enol etherification reaction. By reacting a tyrosine-containing compound with an olefin compound under certain conditions, the olefin compound can locate the phenolic hydroxyl group of the tyrosine-containing compound, react at a fixed point, displace the hydrogen on the phenolic hydroxyl group, and obtain an enol etherified tyrosine compound. The enol etherification reaction method of the tyrosine-containing compound provided by the present invention does not require the introduction of a directing group, has simple operations, mild reaction conditions, and high site selectivity.

[0038] Based on the ene-etherification reaction of tyrosine-containing compounds and olefin compounds, the present invention provides a method for the directed modification of polypeptides or proteins. By performing site-directed ene-etherification on polypeptides or proteins containing tyrosine, directed chemical modification of polypeptides or proteins can be achieved, which can be applied in medicinal chemistry to perform directed chemical modification on polypeptide or protein drugs containing tyrosine, improve the biological activity of the drugs, and enhance their bioavailability; it can also be applied in the labeling of polypeptides or proteins to achieve the tracing of polypeptides or proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the reaction principle diagram of the ene-etherification reaction of a tyrosine-containing compound of the present invention;

[0040] Figure 2 is the fluorescence imaging diagram of the tyrosine site-selective modification of BSA protein in Example 9 of the present invention;

[0041] Figure 3 is the fluorescence baking and staining diagram of the tyrosine site-selective modification of BSA protein in Example 9 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following descriptions are usually only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] The present invention provides a method for the ene-etherification reaction of a tyrosine-containing compound, comprising the following steps: reacting a tyrosine-containing compound with an olefin compound in a solvent under the action of a catalyst, an oxidant, an additive, and a base to obtain an ene-etherified tyrosine compound.

[0044] For example, when the tyrosine-containing compound is and the olefin compound is the above reaction occurs under the action of the catalyst palladium acetate, the oxidant potassium persulfate, the additive TBAB, and the base KHCO3. The reaction principle refers to Figure 1 , which is the reaction principle diagram of the ene-etherification reaction of a tyrosine-containing compound. The reaction principle is as follows: under the action of TBAB and KHCO3, the phenolic hydroxyl group of the Tyr side chain of the tyrosine-containing compound is transformed into a negative ion to form an intermediate compound I. Subsequently, palladium oxidation inserts and coordinates with the phenolic hydroxyl group to form an intermediate II. Then, the olefin compound adds and inserts with palladium to form a palladium-olefin complex III. Subsequently, addition migration occurs to form a new complex IV. Finally, reductive elimination gives the product ene-etherified tyrosine compound.

[0045] Example 1

[0046] The structural formula of the enol-etherified tyrosine compound is as follows: Its preparation is carried out according to the following method: Weigh 0.2 mmol of ethyl acetyl-L-tyrosinate and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl perbenzoate, 0.6 mmol of ethyl acrylate, and react at 20 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated under vacuum. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate. Take the organic phase, dry it with anhydrous Na2SO4, concentrate it under vacuum to obtain a crude product, and purify it by column chromatography to obtain 29.5 mg of the product, namely the enol-etherified tyrosine compound.

[0047] Take the product enol-etherified tyrosine compound for NMR detection, and the NMR data are as follows: 1 H NMR(400MHz,CDCl3)δ7.78(d,J=12.2Hz,1H),7.17–7.09(m,2H),7.06–6.96(m,2H),6.03(d,J=7.8Hz,1H),5.56(d,J=12.2Hz,1H),4.86(dt,J=7.8,5.8Hz,1H),4.19(pd,J=6.5,5.9,3.8Hz,4H),3.13(qd,J=14.0,5.8Hz,2H),2.02(s,3H),1.28(dt,J=11.6,7.1Hz,6H). 13 C NMR(101MHz,CDCl3)δ171.54,169.65,167.23,158.86,154.98,132.74,130.84,118.05,102.29,61.67,60.14,53.18,37.23,23.17,14.32,14.15.

[0048] It can be proved by the NMR data analysis of the product that the enol-etherified tyrosine compound (Ⅰ-1) is obtained by the method of this example.

[0049] Example 2

[0050] The structural formula of the enol-etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0051] 0.2 mmol of tert-butoxycarbonyl-L-tyrosine methyl ester was weighed and added to 1.5 mL of dichloromethane solvent. Then, 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl perbenzoate, 0.6 mmol of ethylene glycol diacrylate were added thereto, and the reaction was carried out at 30 °C for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase was collected and concentrated under vacuum. 10 mL of ethyl acetate and 5 mL of water were added to a separatory funnel, shaken, mixed well, allowed to stand, and separated. The organic phase was taken, dried over anhydrous Na2SO4, concentrated under vacuum to obtain a crude product, which was purified by column chromatography to obtain a product, namely, the enol etherified tyrosine compound, 16.7 mg.

[0052] The obtained product, the enol etherified tyrosine compound, was subjected to NMR detection, and the NMR data were as follows: 1 H NMR(500MHz,Chloroform-d)δ7.81(d,J=12.2Hz,1H),7.17–7.11(m,2H),7.02–6.98(m,2H),6.98–6.94(m,5H),6.75(d,J=8.1Hz,5H),6.55(s,2H),6.44(dd,J=17.3,1.4Hz,1H),6.15(dd,J=17.3,10.5Hz,1H),5.87(dd,J=10.5,1.4Hz,1H),5.57(d,J=12.2Hz,1H),5.06(t,J=9.0Hz,3H),4.58–4.51(m,3H),4.40(s,4H),3.72(d,J=5.3Hz,11H),3.00(qd,J=13.7,5.7Hz,6H),1.42(s,28H).

[0053] 13 C NMR(126MHz,CDCl3)δ172.64,172.20,167.05,166.04,159.70,155.30,154.83,132.99,131.46,130.84,130.33,127.97,127.36,118.07,115.52,101.46,80.15,62.39,61.88,54.63,54.42,52.33,52.23,50.72,37.69,37.52,28.29.

[0054] It can be proved by the NMR data analysis of the product that the enol etherified tyrosine compound (Ⅰ-11) was obtained by the method of this example.

[0055] Example 3

[0056] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0057] (I-13) The structures of the two substrates used in the synthesis are shown as follows:

[0058]

[0059] Weigh 0.1 mmol of (I-13a) and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl peroxybenzoate, 0.6 mmol of (I-13b), and react at 37 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated under vacuum. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate. Take the organic phase, dry it with anhydrous Na2SO4, concentrate it under vacuum to obtain the crude product, and purify it by column chromatography to obtain the product, that is, 43.7 mg of the enol etherified tyrosine compound.

[0060] Take the product enol etherified tyrosine compound for NMR detection, and the NMR data are as follows: 1 H NMR(500MHz,Chloroform-d)δ7.78(d,J=12.2Hz,1H),7.13(d,J=8.6Hz,2H),6.99(d,J=8.6Hz,2H),6.03(d,J=7.7Hz,1H),5.51(d,J=12.2Hz,1H),5.35(d,J=8.4Hz,1H),4.89–4.80(m,1H),4.63–4.48(m,2H),4.40(dd,J=11.2,3.5Hz,1H),4.18(td,J=7.1,3.1Hz,2H),3.77(s,3H),3.17–3.07(m,2H),2.01(s,3H),1.45(s,9H),1.25(d,J=7.1Hz,3H).

[0061] 13 C NMR(126MHz,CDCl3)δ171.50,169.62,166.65,159.86,154.75,133.03,130.88,130.27,121.60,118.07,101.11,80.34,64.02,61.65,53.16,52.72,37.25,28.27,23.15,21.10,14.14.

[0062] It can be proved by the NMR data analysis of the product that the method of this example obtained the enol etherified tyrosine compound (Ⅰ-13).

[0063] Example 4

[0064] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0065] (I-16) The structures of the two substrates used in the synthesis are shown as follows:

[0066]

[0067] Weigh 0.1 mmol of (I-16a) and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl peroxybenzoate, 0.6 mmol of (I-16b), and react at 37 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated under vacuum. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate the layers. Take the organic phase, dry it with anhydrous Na2SO4, concentrate it under vacuum to obtain the crude product, and purify it by column chromatography to obtain 43.9 mg of the product, that is, the enol etherified tyrosine compound.

[0068] Take the product enol etherified tyrosine compound for NMR detection, and the NMR data are as follows: 1 H NMR(500MHz,Chloroform-d)δ7.79(d,J=12.2Hz,1H),7.15(d,J=8.1Hz,2H),7.03–6.99(m,2H),6.97(d,J=7.9Hz,1H),5.51(d,J=12.2Hz,1H),5.04(dd,J=7.8,5.3Hz,2H),4.86(dt,J=7.9,3.8Hz,1H),4.58(q,J=6.6Hz,1H),4.48(ddd,J=42.7,11.4,3.9Hz,2H),4.23(s,1H),3.78(s,3H),3.73(s,3H),3.08(ddd,J=47.8,13.9,6.0Hz,2H),1.44(s,9H),1.43(s,9H),1.38(d,J=7.1Hz,3H).

[0069] 1313C NMR (126 MHz, CDCl3) δ 172.60, 172.14, 169.71, 166.73, 160.00, 155.02, 154.75, 133.26, 133.12, 130.88, 130.06, 128.37, 118.02, 100.98, 80.07, 63.46, 54.39, 52.84, 52.30, 51.85, 38.62, 37.70, 28.28, 18.08.

[0070] The NMR data analysis of the product can prove that the method of this example obtains the enol etherified tyrosine compound (Ⅰ-16).

[0071] Example 5

[0072] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0073] (I-18) The substrate used for synthesis has the following structure:

[0074]

[0075] Weigh 0.1 mmol of (I-18a) and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl perbenzoate, 0.6 mmol of (I-13b), and react at 55 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated under vacuum. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate. Take the organic phase, dry it with anhydrous Na2SO4, concentrate it under vacuum to obtain the crude product, and purify it by column chromatography to obtain 43.7 mg of the product, that is, the enol etherified tyrosine compound.

[0076] Take the product enol etherified tyrosine compound for nuclear magnetic detection, and the NMR data are as follows: 11H NMR (500 MHz, Chloroform-d) δ 7.78 (d, J = 12.2 Hz, 1H), 7.23–7.21 (m, 2H), 7.00–6.96 (m, 2H), 6.61–6.49 (m, 2H), 6.42 (dd, J = 8.1, 3.2 Hz, 1H), 5.52 (d, J = 12.2 Hz, 1H), 5.38 (d, J = 8.6 Hz, 1H), 4.75–4.72 (m, 1H), 4.61–4.51 (m, 1H), 4.42 (dd, J = 8.7, 5.0 Hz, 2H), 3.77 (s, 3H), 3.71 (s, 3H), 3.06 (dd, J = 6.9, 3.7 Hz, 2H), 2.14–2.10 (m, 1H), 1.99 (s, 3H), 1.45 (s, 9H), 0.89–0.86 (m, 6H).

[0077] 13 13C NMR (126 MHz, CDCl3) δ 171.68, 170.79, 170.22, 166.70, 159.97, 154.71, 133.40, 130.89, 130.33, 121.68, 118.18, 115.58, 101.04, 80.34, 57.50, 54.45, 52.72, 52.17, 37.62, 31.11, 28.28, 23.07, 21.09, 18.83, 17.71.

[0078] It can be proved by the NMR data analysis of the product that the method of this example obtained the enol etherified tyrosine compound (I-18).

[0079] Example 6

[0080] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0081] (I-28) The substrate used in the synthesis has the following structure:

[0082]

[0083] Weigh 0.1 mmol (I-28a) and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium(II) trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl peroxybenzoate, 0.6 mmol of (I-28b), and react at 40 °C for 24 h. After the reaction is completed, cool the reaction solution to room temperature, filter it through diatomaceous earth, wash it 3 times with dichloromethane, collect the organic phase, and concentrate it under vacuum. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let it stand, and separate the layers. Take the organic phase, dry it with anhydrous Na2SO4, concentrate it under vacuum to obtain the crude product, and purify it by column chromatography to obtain the product, namely the enol etherified tyrosine compound, 46.9 mg.

[0084] Take the enol etherified tyrosine compound as the product for NMR detection. The NMR data are as follows: 1 H NMR(500MHz,Chloroform-d)δ7.92(d,J=12.2Hz,1H),7.24–7.21(m,2H),7.15(d,J=8.1Hz,1H),7.06–7.03(m,2H),6.94(d,J=8.0Hz,2H),6.75(d,J=7.9Hz,2H),6.55–6.51(m,1H),5.70(d,J=12.1Hz,1H),5.08(s,1H),4.83(s,1H),4.47(dd,J=8.6,5.1Hz,1H),4.39–4.31(m,1H),4.30–4.21(m,1H),3.73(s,3H),3.71(s,3H),3.33(tt,J=7.2,3.7Hz,2H),3.20(dd,J=14.0,5.7Hz,1H),3.14–3.09(m,1H),3.02(dd,J=13.9,6.8Hz,1H),2.95–2.88(m,1H),2.15–2.09(m,1H),1.81(d,J=23.6Hz,4H),1.43(s,18H),0.87(dd,J=14.5,6.9Hz,6H). 13CNMR(126MHz,CDCl3)δ171.77,171.50,171.13,165.53,160.57,155.49,154.76,149.59,134.02,130.84,130.29,130.20,121.82,118.12,118.09,115.52,115.44,101.31,80.95,80.43,57.31,55.77,53.41,52.38,52.26,52.10,46.96,37.36,37.12,31.22,29.67,28.25,18.82,17.74.

[0085] It can be proved by the NMR data analysis of the product that the method of this example obtained the enol etherified tyrosine compound (I-28).

[0086] Example 7

[0087] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out according to the following method:

[0088] (I-30) The substrate used for synthesis has the following structure:

[0089]

[0090] Weigh 0.1 mmol of (I-30a) and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl peroxybenzoate, 0.6 mmol of (I-30b), and react at 37 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated in vacuo. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate. Take the organic phase, dry it with anhydrous Na2SO4, concentrate in vacuo to obtain the crude product, and purify it by column chromatography to obtain 45.1 mg of the product, that is, the enol etherified tyrosine compound.

[0091] Take the product enol etherified tyrosine compound for NMR detection, and the NMR data are as follows: 11H NMR (500 MHz, Chloroform-d) δ 7.94 (d, J = 12.2 Hz, 1H), 7.14 (s, 2H), 7.06–7.04 (m, 2H), 7.03–7.00 (m, 2H), 6.91 (d, J = 8.1 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 6.36 (d, J = 7.8 Hz, 2H), 5.69 (d, J = 12.1 Hz, 1H), 4.84 (ddt, J = 19.3, 7.9, 5.8 Hz, 2H), 3.86 (dd, J = 9.7, 4.2 Hz, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 3.19–3.05 (m, 4H), 1.45 (d, J = 1.3 Hz, 18H), 0.95 (d, J = 2.8 Hz, 18H). 13 13C NMR (126 MHz, CDCl3) δ 172.29, 171.71, 171.48, 170.67, 165.58, 160.64, 155.58, 154.80, 149.66, 130.94, 130.31, 130.21, 121.76, 118.22, 115.62, 101.30, 80.06, 79.95, 62.44, 62.25, 54.40, 53.29, 53.09, 52.42, 52.24, 37.68, 37.21, 36.90, 34.50, 34.36, 28.33, 26.44, 26.39.

[0092] Analysis of the NMR data of the product can prove that the method of this example obtained the enol etherified tyrosine compound (Ⅰ-30).

[0093] Example 8

[0094] The structural formula of the enol etherified tyrosine compound is as follows: Its preparation is carried out as follows: Weigh 0.1 mmol of Boc-Tyr-OMe and add it to 1.5 mL of dichloromethane solvent. Then add 0.01 mmol of palladium trifluoroacetate, 0.3 mmol of tetrabutylammonium bromide, 0.1 mmol of potassium carbonate, 0.3 mmol of tert-butyl peroxybenzoate, 0.6 mmol of menthol, and react at 37 °C for 24 h. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through diatomaceous earth, washed 3 times with dichloromethane, the organic phase is collected and concentrated in vacuo. Add 10 mL of ethyl acetate and 5 mL of water to a separatory funnel, shake, mix well, let stand, and separate. Take the organic phase, dry it with anhydrous Na2SO4, and concentrate in vacuo to obtain the crude product. The product is purified by column chromatography to obtain 38.2 mg of the product, that is, the enol etherified tyrosine compound.

[0095] The product enol etherified tyrosine compound was subjected to NMR detection, and the NMR data are as follows: 1 H NMR(500MHz,Chloroform-d)δ7.76(d,J=12.2Hz,1H),7.14(d,J=8.4Hz,2H),7.01(d,J=8.6Hz,2H),5.54(d,J=12.2Hz,1H),5.01(d,J=8.3Hz,1H),4.77(td,J=10.9,4.4Hz,1H),4.59(q,J=6.6Hz,1H),3.73(s,3H),3.08(ddd,J=50.3,13.9,6.0Hz,2H),2.03(dtd,J=12.0,3.7,1.8Hz,1H),1.89(pd,J=7.0,2.8Hz,1H),1.72–1.67(m,2H),1.52(tt,J=6.7,3.3Hz,1H),1.43(s,9H),1.41–1.37(m,2H),1.10–0.98(m,2H),0.91(dd,J=6.8,5.6Hz,6H),0.79(d,J=6.9Hz,3H).

[0096] 13 C NMR(126MHz,CDCl3)δ172.17,166.81,158.76,154.95,132.80,130.80,118.14,102.57,80.06,73.80,54.39,52.30,47.15,41.07,37.72,34.29,31.40,29.69,28.29,26.36,23.57,22.03,20.74,16.47.

[0097] Analysis of the NMR data of the product demonstrated that the enol etherified tyrosine compound (Ⅰ-31) was obtained by the method of this example.

[0098] Example 9 Application of tyrosine C(sp2)-O enol etherification in protein modification Using bovine serum albumin (BSA) as the modified protein, acrylated BODIPY as the labeling substrate, palladium trifluoroacetate as the catalyst, and MeCN solution (volume ratio: MeCN:H2O = 1:3) as the solvent, BSA fluorescence labeling was carried out. The reaction equation is as follows:

[0099]

[0100] It was carried out in the following steps:

[0101] Weigh 1 mg of BSA (1 eq) and 500 μg of acrylated BODIPY and dissolve them in 4.5 mL of PBS with a pH of 8.4. Mix well to obtain a PBS mixture; weigh palladium trifluoroacetate (0.1 eq), TBAB (2 eq), and TBPB (1.5 eq) and dissolve them in MeCN. Mix well to obtain a MeCN mixture; respectively take 900 μL of the PBS mixture and 300 μL of the MeCN mixture and mix them well in a 2.5 mL ep tube. Place the mixed reaction tube in a metal bath reactor and shake overnight at a reaction temperature of 37 °C. Take the obtained reaction solution and add it to a dialysis bag (MW: 35000), and place it in 10× neutral PBS for dialysis 3 times, with each dialysis lasting 2 h. Take 20 μL of the dialyzed protein and add it to an ep tube, add 2.5 μL of loading buffer, and boil it at 95 °C for 10 min. Then take 10 μL and add it to the sample loading well, connect to the power supply at 90 V for 1 h, then adjust the voltage to 120 V and keep it for 0.5 h. After that, remove the gel plate. Place the gel plate in the fluorescence channel to observe the labeling situation, and finally perform baking and staining. The fluorescence imaging map and baking and staining map of tyrosine site-selective modification of BSA protein are shown in Figure 2 and Figure 3 .

[0102] The results of this example show that this reaction can successfully label BSA. At the same time, in the control group without adding a catalyst, there is only very weak fluorescence labeling. We set three groups with mass ratios of protein to fluorescent dye of 15:1, 10:1, and 5:1 respectively. The results show that the labeling effects are similar and there are no significant differences. The number of tyrosine residues contained in the protein is limited, that is, the labeling results are similar or the same.

[0103] Comparative Example 1

[0104] The main difference from Example 1 is that the catalyst is palladium tris(triphenylphosphine). Other operating steps are the same as those in Example 1.

[0105] The reaction result is: The product obtained after column chromatography purification is 11.8 mg.

[0106] Comparative Example 2

[0107] The main difference from Example 1 is that the catalyst is dichlorotriphenylphosphine palladium. Others are the same as in Example 1.

[0108] The reaction result is: The product obtained after column chromatography purification is 8.68 mg.

[0109] Comparative Example 3

[0110] The main difference from Example 1 is that the catalyst is DPPF palladium dichloride. Others are the same as in Example 1.

[0111] The reaction result was: The product obtained after column chromatography purification was 6.6 mg.

[0112] Combining the reaction results of Example 1 and Comparative Examples 1-3, it can be thus illustrated that when the reaction occurs under the catalysis of other catalysts, such as tetrakis(triphenylphosphine)palladium, dichlorotriphenylphosphine palladium, and DPPF dichloropalladium, the reaction yield is relatively low. In the enol etherification reaction of the present invention, the catalysts are only palladium acetate and palladium trifluoroacetate, and the enol etherification reaction of the present invention has a better effect.

[0113] Comparative Example 4

[0114] The main difference from Example 1 was: The oxidant was oxygen. Others were the same as in Example 1.

[0115] The reaction result was: The product obtained after column chromatography purification was 6.35 mg.

[0116] Comparative Example 5

[0117] The main difference from Example 1 was: The oxidant was hydrogen peroxide. Others were the same as in Example 1.

[0118] The reaction result was: The product obtained after column chromatography purification was 6.78 mg.

[0119] Comparative Example 6

[0120] The main difference from Example 1 was: The oxidant was tert-butyl hydroperoxide. Others were the same as in Example 1.

[0121] The reaction result was: The product obtained after column chromatography purification was 18.73 mg.

[0122] Comparative Example 7

[0123] The main difference from Example 1 was: The oxidant was copper acetate. Others were the same as in Example 1.

[0124] The reaction result was: Basically no reaction occurred.

[0125] Comparative Example 8

[0126] The main difference from Example 1 was: The oxidant was silver acetate. Others were the same as in Example 1.

[0127] The reaction result was: Basically no reaction occurred.

[0128] Combined with the reaction results of Example 1 and Comparative Examples 4-8, it is thus shown that when adding other oxidants to promote the reaction, such as oxygen, hydrogen peroxide, tert-butyl hydroperoxide, copper acetate, and silver acetate, the reaction yield is relatively low. Among them, when adding copper acetate or silver acetate as the oxidant, the reaction hardly occurs. In the enol etherification reaction of the present invention, the oxidant is selected from potassium persulfate, silver acetate, and tert-butyl peroxybenzoate, and the enol etherification reaction of the present invention has a better effect.

[0129] Comparative Example 9

[0130] The main difference from Example 1 is that the reaction temperature is 15°C. Others are the same as in Example 1.

[0131] The reaction result is that the product obtained after column chromatography purification is 19.91 mg.

[0132] Comparative Example 9

[0133] The main difference from Example 1 is that the reaction temperature is 60°C. Others are the same as in Example 1.

[0134] The reaction result is that the product obtained after column chromatography purification is 20.01 mg.

[0135] From the comparison of the reaction results of Example 1 and Comparative Examples 8-9, it can be seen that when the reaction temperature of the enol etherification reaction is 15°C or 60°C, the reaction yield is relatively low. Combining with other examples, it can be shown that in the enol etherification reaction of the present invention, the reaction temperature is preferably 20-55°C, and the reaction has a better effect at this reaction temperature.

[0136] The method for column chromatography purification in the examples of the present invention is as follows: a mixed solution with a volume ratio of ethyl acetate to petroleum ether of 1:10 is used as the mobile phase, and the eluate with an Rf value of 0.3-0.5 is collected by TLC tracking. The collected eluate is concentrated under reduced pressure to remove the solvent and dried to obtain the product. At the same time, through verification in the present invention, when the mobile phase is a mixed solution with a volume ratio of ethyl acetate to petroleum ether of 1:8-10, good elution effects can be obtained.

[0137] The reactions in the examples of the present invention are all carried out in a pressure-resistant tube under a closed condition. Therefore, when the reaction temperature is higher than the boiling point of the solvent, the reaction can be carried out at this temperature.

[0138] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0139] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for the enol etherification reaction of a tyrosine-containing compound, characterized in that: It includes the following steps: In a solvent, a tyrosine-containing compound with the structural general formula (Ⅱ) reacts with an olefin compound with the structural general formula (Ⅲ) under the action of a catalyst, an oxidant, an additive, and a base to obtain an enol etherified tyrosine compound with the structural general formula (Ⅰ): ； Wherein, R1 is selected from a straight-chain or branched-chain alkoxy group with 1 to 7 carbon atoms or a straight-chain or branched-chain alkoxycarbonyl group with 1 to 7 carbon atoms; R2 is selected from a straight-chain or branched-chain alkyl group with 1 to 7 carbon atoms; R3 is selected from a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a phenoxycarbonyl group, a chlorobenzene, or a tert-butyl ether; Wherein, the catalyst is palladium acetate and / or palladium trifluoroacetate; the oxidant is selected from one or more of potassium persulfate, silver acetate, and tert-butyl peroxybenzoate.

2. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: R1 is an acetyl group or a tert-butoxycarbonyl group, R2 is a methyl group or an ethyl group; R3 is selected from a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a phenoxycarbonyl group, a p-methylphenoxycarbonyl group, a chlorobenzene, or a tert-butyl ether.

3. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: The molar ratio of the tyrosine-containing compound, the olefin compound, the catalyst, the oxidant, the additive, and the base is 1:1~3:0.1~0.3:1~3:1~2:0.5~1.

2.

4. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: The additive is selected from one or more of tetrabutylammonium bromide, tetrabutylammonium iodide, and tetrabutylammonium chloride.

5. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: The base is selected from one or more of potassium carbonate, cesium carbonate, potassium fluoride, and cesium fluoride.

6. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: The solvent is dichloromethane.

7. The method for the enol etherification reaction of a tyrosine-containing compound according to claim 1, characterized in that: After the reaction, purification is carried out. The purification method includes the following steps: (1) Take the reaction product for diatomaceous earth filtration, then wash it with DCM, then collect the organic phase, concentrate it, extract it with ethyl acetate and water, take the organic layer, dry it, filter it, and concentrate it to obtain a crude product; (2) Take the crude product for silica gel column chromatography, use a mixed solution of ethyl acetate and petroleum ether as the mobile phase, collect the eluate with an Rf value of 0.3~0.5, and remove the solvent from the eluate to obtain the enol etherified tyrosine compound.

8. A method for the directed modification of a polypeptide or protein, characterized in that: It includes the following steps: Based on the enol etherification reaction method described in any one of claims 1-7, chemically modify a polypeptide or a protein; Wherein, the polypeptide or the protein contains tyrosine.

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