3-acyl-4-pyrone derivatives as coupling reagents and their use in the preparation of polypeptide, protein conjugates

Abstract

The application belongs to the technical field of organic synthesis and protein modification, and particularly relates to a 3-acyl-4-pyrone derivative as a coupling reagent and application thereof in preparation of polypeptide and protein conjugates. The 3-acyl-4-pyrone derivative can rapidly and efficiently chemoselectively undergo O to N insertion substitution reaction with a lysine residue in a polypeptide or protein sequence under near physiological conditions, so as to realize coupling of the polypeptide and the protein. The 3-acyl-4-pyrone derivative has a simple synthesis method, simple and convenient modification operation for the protein, novel structure, and has not been reported to be applied to polypeptide and protein modification. The 3-acyl-4-pyrone derivative provides a new technology and a new way for various functional modification of polypeptides and proteins.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic synthesis and protein modification, and particularly relates to a 3-acyl-4-pyrone derivative as a coupling reagent and application thereof in preparation of polypeptide and protein conjugates. BACKGROUND

[0002] Chemical modification of polypeptides or proteins is an important method for improving their physicochemical properties, pharmacokinetic properties and biological activities, such as increasing stability, prolonging in vivo action time, increasing therapeutic effect and reducing toxic side effects, etc. By introducing specific functional groups such as directing groups, fluorophores, drugs, etc., the distribution of polypeptides or proteins in the life system can be changed, biomedical imaging and disease treatment can be performed, etc. Generally, coupling reagents need to react with some functional groups in polypeptides or proteins. Since lysine accounts for 5.9% of human proteome and is usually exposed on the surface of proteins, lysine is an important modification site.

[0003] The classical modification method of lysine residues in polypeptides or proteins is to use pre-prepared N-hydroxysuccinimide (NHS) ester to form a stable amide bond with lysine residues. However, NHS ester is easily hydrolyzed in aqueous solution, and can react with tyrosine, serine and threonine to generate byproducts, and chemical modification often leads to a decrease in protein activity, which limits its application. Therefore, it is urgent to develop a new lysine coupling method to achieve efficient modification of polypeptides and proteins. SUMMARY

[0004] The present application solves the above technical problems existing in the prior art and provides a polypeptide and protein coupling reagent containing a 3-substituted-4-pyrone core structure. The structure core is obtained by simplifying the natural product azaphilone. It can quickly and efficiently undergo O→N insertion substitution reaction with lysine residues in polypeptide or protein sequences under near-physiological conditions, realizing the coupling of polypeptides and proteins.

[0005] The technical scheme of the present application is as follows:

[0006] A 3-acyl-4-pyrone derivative, comprising a compound of general formula (I) or a chemically acceptable salt thereof;

[0007]

[0008] wherein R is selected from carboxyl, amine, hydroxyl, alkynyl, azido, thiol, succinimide or maleimide.

[0009] Preferably, R is selected from

[0010]

[0011] Preferably, said chemically acceptable salt is an addition salt of said compound of general formula (I) with an acid selected from hydrogen chloride, hydrogen bromide, trifluoroacetic acid, sulfuric acid, p-toluensulfonic acid, benzenesulfonic acid, methanesulfonic acid, acetic acid or phosphoric acid.

[0012] Preferably, said R groups are used as derivatization sites for the introduction of a modifying group. More preferably, said modifying group is selected from a fluorophore, a chemically and biologically functional molecule or a modifying group.

[0013] A first method for the preparation of 3-acyl-4-pyrone derivatives, the synthetic route being as follows:

[0014]

[0015] wherein R 1 represents

[0016] Preferably, said method comprises the steps of using compound II as the initial material, obtaining compound III by acid catalyzed hydrolysis, and obtaining compound IV by acylation with an amine after reaction with oxalyl chloride and thionyl chloride.

[0017] Preferably, said method comprises the steps of dissolving compound II in dichloromethane, adding trifluoroacetic acid and triethylsilane dropwise under nitrogen protection at -15°C, spinning dry the solution after the reaction is complete to obtain a white solid, dissolving the white solid in dichloromethane again under nitrogen protection at 0°C, then adding oxalyl chloride and DMF dropwise, stirring at 0°C for 2 hours, spinning dry after the reaction is complete, dissolving in dichloromethane, and stirring at 0°C, slowly adding an ethanolamine solution in dichloromethane, and stirring while maintaining the temperature to obtain the product.

[0018] A second method for the preparation of 3-acyl-4-pyrone derivatives, the synthetic route being as follows:

[0019]

[0020] wherein R 2 represents

[0021] R 3 represents

[0022] Preferably, said method comprises the steps of using compound II as the initial material, obtaining compound III by acid catalyzed hydrolysis, obtaining compound V by esterification with an alcohol, and obtaining compound VI by acid catalyzed deprotection.

[0023] Preferably, the preparation method comprises the following steps: dissolving compound II in 4.0 mL of dichloromethane, adding trifluoroacetic acid and triethylsilane dropwise under nitrogen protection at -15 ℃; after the reaction is completed, spin-drying to obtain a white solid; dissolving the white solid and alcohol in dichloromethane under nitrogen protection, stirring at 0 ℃; dissolving EDCI and DMAP in dichloromethane, and adding into the reaction system, and reacting at 24 ℃ for 18 h to obtain the product.

[0024] The third preparation method of the 3-acyl-4-pyrone derivative is as follows:

[0025]

[0026] wherein R 4 represents R 5 represents

[0027] Preferably, the preparation method comprises the following steps: taking compound VII as the initial raw material, reacting with aldehyde under the action of a base to obtain VIII, and reacting with an oxidant to obtain compound IX, and then performing a click chemistry reaction to obtain X.

[0028] Preferably, the preparation method comprises the following steps: dissolving compound VII in anhydrous methanol under nitrogen protection, and stirring at 0 ℃; dissolving sodium methoxide in methanol, and adding into the reaction system, followed by adding aldehyde, and reacting at 23 ℃ for 10 h; after the reaction is completed, adding glacial acetic acid to quench, stirring, and spin-drying to obtain a gray solid; dissolving the gray solid in dichloromethane, adding DMP, and reacting at 23 ℃ for 3 h to obtain the product.

[0029] The 3-acyl-4-pyrone derivative can be used as a polypeptide and protein coupling reagent. The coupling reagent can quickly and efficiently react with lysine in a polypeptide or protein sequence, so as to realize the coupling of the polypeptide and the protein.

[0030] The method for coupling a polypeptide or a protein by using the 3-acyl-4-pyrone derivative comprises the following steps: coupling a primary amine in a polypeptide or a protein with a compound of formula (I) in water, a pH buffer, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide or a mixture thereof, at a reaction pH of 6.5-10.0 and a reaction temperature of 0-40 ℃, to obtain a polypeptide coupling product as shown in formula (X) or a protein coupling product as shown in formula (XI).

[0031]

[0032] wherein m represents the number of free amine groups in the protein, and m = 1-7.

[0033] Preferably, the polypeptide is polypeptide sequence 1 (AcNH-DRVYIHGKF-CONH2), polypeptide sequence 2 (AcNH-QAWECMKF-CONH2) or polypeptide sequence 3 (AcNH-APSKF-CONH2); and the protein is lysozyme (Lysozyme C).

[0034] The advantages of the present application over the prior art are as follows,

[0035] The present application simplifies the structure of natural product azafenidin, and the obtained polypeptide and protein coupling reagent containing 3-substituted-4-pyrone structure are novel in structure, high in solubility, simple and convenient in modification operation of proteins, rapid in reaction, and have not been reported to be applied to polypeptide and protein modification, thereby providing a new technology and new way for various functional modification of polypeptides and proteins. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Secondary mass spectrum of polypeptide coupling product P-1;

[0037] Figure 2 Secondary mass spectrum of polypeptide coupling product P-2;

[0038] Figure 3 Secondary mass spectrum of polypeptide coupling product P-3;

[0039] Figure 4 Mass spectrum (maldi-tof) of lysozyme (lysozyme C) coupling product. DETAILED DESCRIPTION

[0040]

[0041] Compound II was synthesized by the method reported in the patent (CN113444102A).

[0042] Example 1:

[0043]

[0044] Compound II 50 mg (0.25 mmol, 1.0 eq) was dissolved in 4.0 mL of dichloromethane, and trifluoroacetic acid 0.38 mL (5.1 mmol, 20 eq) and triethylsilane 0.41 mL (2.5 mmol, 10 eq) were added dropwise under nitrogen protection at -15 °C. The reaction was monitored by TLC, and the solution was dried to obtain 35 mg of white solid. The white solid 100 mg (0.71 mmol, 1.0 eq) was added to the reaction bottle, and dichloromethane 30 mL was added under nitrogen protection at 0 °C, followed by dropwise addition of oxalyl chloride 135 mg (0.11 mmol, 1.5 eq), and one drop of DMF. After addition, the mixture was stirred at 0 °C for 2 h. The reaction was monitored by TLC, and the solution was dried and dissolved in dichloromethane 5 mL, and stirred at 0 °C. Ethanolamine 65 mg (0.10 mmol, 1.4 eq) in dichloromethane was added dropwise slowly, and the temperature was maintained after addition. The reaction was monitored by TLC, and the solution was dried and purified by silica gel column chromatography (dichloromethane:methanol = 80:1) to obtain 42 mg of oil, 32%.

[0045] NMR and mass spectrometry characterization of IV-1: 1 H NMR (400 MHz, Chloroform-d) δ 9.53 (s, 1H), 8.76 (d, J = 1.1 Hz, 1H), 7.82 (dd, J = 5.7, 1.1 Hz, 1H), 6.50 (d, J = 5.7 Hz, 1H), 3.76 (s, 2H), 3.56 (q, J = 5.4 Hz, 2H), 3.30 (brs, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 177.74, 163.49, 161.89, 155.63, 120.66, 118.93, 62.28, 42.55. HRMS (ESI, m / z): [M+Na] + calcd for C8H9NNaO4 + 184.0604; found 184.0603.

[0046] Example 2:

[0047] The method of Example 1 was used, when R 1 represents Compound IV-2 was prepared.

[0048]

[0049] NMR and mass spectrometry characterization of IV-2: 1H NMR (400 MHz, Chloroform-d) δ 9.49 (s, 1H), 8.80 (d, J = 1.1 Hz, 1H), 7.83 (dd, J = 5.7, 1.1 Hz, 1H), 6.54 (d, J = 5.7 Hz, 1H), 4.20 (dd, J = 5.5, 2.6 Hz, 2H), 2.24 (t, J = 2.5 Hz, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 177.60, 169.34, 162.66, 161.93, 155.55, 120.57, 119.11, 61.57, 41.45, 14.28. HRMS (ESI, m / z): [M+Na] + calcd for C 10 H 11 NNaO5 + 248.0529; found 248.0526.

[0050] Example 3:

[0051] The compound IV-3 was prepared in the same manner as in Example 1, when R 1 represents The compound IV-3 was prepared in the same manner as in Example 1, when R

[0052]

[0053] NMR and mass spectrum characterization of IV-3: 1 H NMR (400 MHz, Chloroform-d) δ 9.49 (s, 1H), 8.80 (d, J = 1.1 Hz, 1H), 7.83 (dd, J = 5.7, 1.1 Hz, 1H), 6.54 (d, J = 5.7 Hz, 1H), 4.20 (dd, J = 5.5, 2.6 Hz, 2H), 2.24 (t, J = 2.5 Hz, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 177.59, 162.15, 161.99, 155.60, 120.61, 119.09, 79.34, 71.51, 28.93. HRMS (ESI, m / z): [M+Na] + calcd for C9H7NNaO3 + 178.0499; found 178.0498.

[0054] Example 4:

[0055]

[0056] Compound 7 50 mg (0.25 mmol, 1.0 eq) was dissolved in 4.0 mL of dichloromethane, and trifluoroacetic acid 0.38 mL (5.1 mmol, 20 eq) and triethylsilane 0.41 mL (2.5 mmol, 10 eq) were added dropwise at -15 °C under nitrogen protection. The reaction was monitored by TLC, and the reaction was completed. The solution was rotary evaporated to get white solid 35 mg. White solid 49 mg (0.35 mmol, 2.0 eq) and alcohol 43 mg (0.18 mmol, 1.0 eq) were added into the reaction bottle, and dissolved in 5 mL of dichloromethane under nitrogen protection. EDCI 67 mg (0.35 mmol, 2.0 eq) and DMAP 1 mg (0.008 mmol, 0.1 eq) were dissolved in 1 mL of dichloromethane, and added dropwise into the reaction system. After the addition was completed, the reaction was carried out at 24 °C for 18 h. The reaction was monitored by TLC, and the reaction was completed. The reaction solution was diluted with dichloromethane and washed with saturated sodium chloride once, and dried over anhydrous sodium sulfate. Silica gel column chromatography (dichloromethane:methanol = 60:1) gave oil 24 mg, with a yield of 29%

[0057] V-1 NMR and mass spectrometry characterization: 1 H NMR (400 MHz, Chloroform-d) δ 8.53 (d, J = 1.1 Hz, 1H), 7.76 (dd, J = 5.9, 1.1 Hz, 1H), 6.48 (d, J = 5.9 Hz, 1H), 5.40 (tt, J = 6.8, 4.3 Hz, 1H), 4.72 (ddd, J = 11.0, 6.7, 1.5 Hz, 1H), 4.42 (dddd, J = 16.9, 11.1, 5.7, 1.5 Hz, 2H), 4.08 - 4.15 (m, 3H), 3.86 - 3.61 (m, 6H), 3.40 (dd, J = 5.6, 4.4 Hz, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 173.37, 169.35, 161.96, 161.78, 154.49, 120.36, 120.04, 70.86, 70.76, 70.49, 70.04, 65.27, 58.32, 55.18, 50.71. HRMS (ESI, m / z): [M+Na] + calcd for C 15 H 18 N4NaO7 + 389.1068; found 389.1063.

[0058] Example 5:

[0059] The method of Example 4 was used, when R 2 represents R 2 represents Compound V-2 was prepared.

[0060]

[0061] NMR and mass spectrometry characterization of V-2: 1 H NMR (400 MHz, Chloroform-d) δ 8.42 (d, J = 1.1 Hz, 1H), 7.70 (dd, J = 5.9, 1.1 Hz, 1H), 6.44 (d, J = 5.9 Hz, 1H), 5.00 (p, J = 6.2 Hz, 1H), 1.68 (qd, J = 7.5, 6.0 Hz, 4H), 0.94 (t, J = 7.5 Hz, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 173.94, 162.36, 160.53, 154.37, 121.90, 119.96, 78.25, 26.43, 9.67. HRMS (ESI, m / z): [M + Na] + calcd for C 11 H 14 NaO4 + 233.0784; found 233.0780.

[0062] Example 6:

[0063] In the same manner as in Example 4, when R 2 represents Compound V-3 was prepared.

[0064]

[0065] NMR and mass spectrometry characterization of V-3: 1 H NMR (500 MHz, Chloroform-d) δ 8.41 (s, 1H), 7.70 (d, J = 5.9 Hz, 1H), 6.43 (d, J = 5.7 Hz, 1H), 5.03 - 4.94 (m, 1H), 1.95 - 1.86 (m, 2H), 1.80 - 1.70 (m, 2H), 1.60 - 1.49 (m, 3H), 1.46 - 1.35 (m, 2H), 1.34 - 1.22 (m, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 173.98, 161.94, 160.73, 154.33, 122.02, 120.04, 73.99, 31.57, 25.46, 23.69. HRMS (ESI, m / z): [M + Na] + calcd for C 12 H 14NaO4 + 245.0784; found 235.0785.

[0066] Example 7:

[0067] The procedure of Example 4 was followed when R 2 represents Compound IV-3 was prepared.

[0068]

[0069] NMR and mass spectrometry characterization of V-4: 1 H NMR (400 MHz, Chloroform-d) δ 8.38 (d, J = 1.0 Hz, 1H), 7.69 (dd, J = 5.9, 1.1 Hz, 1H), 6.43 (d, J = 5.9 Hz, 1H), 5.36 (tt, J = 6.2, 2.7 Hz, 1H), 1.99 - 1.86 (m, 2H), 1.86 - 1.69 (m, 4H), 1.69 - 1.56 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 173.95, 162.34, 160.70, 154.35, 121.95, 120.02, 78.56, 32.77, 23.84. HRMS (ESI, m / z): [M + Na] + calcd for C 11 H 12 NaO4 + 231.0628; found 231.0623.

[0070] Example 8:

[0071] The procedure of Example 4 was followed when R 3 represents Compound V-5 was prepared. Compound was dissolved in trifluoroacetic acid:dichloromethane = 1:10, stirred at 0 °C for 3 h, rotary evaporation to give red oil VI-1.

[0072]

[0073] NMR and mass spectrometry characterization of V-4: 1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.63 (s, 2H), 8.23 (d, J = 5.5 Hz, 1H), 6.46 (d, J = 5.8 Hz, 1H), 5.13 (tt, J = 6.5, 3.3 Hz, 1H), 3.31 - 3.21 (m, 2H), 3.19 - 3.10 (m, 2H), 2.07 - 1.96 (m, 2H), 1.92 - 1.82 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 173.60, 162.45, 162.00, 158.87, 158.60, 156.85, 121.20, 119.19, 67.23, 40.44, 27.19. HRMS (ESI, m / z): [M+H] + calcd for C 11 H 14 NO4 + 224.0917; found 224.0915.

[0074] Example 9:

[0075] The compound VI-2 was prepared according to the procedure of Example 8, when R 3 represents The compound VI-2 was prepared according to the procedure of Example 8, when R

[0076]

[0077] VI-2 NMR and Mass Characterization: 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.63 (s, 2H), 8.23 (d, J = 5.5 Hz, 1H), 6.46 (d, J = 5.8 Hz, 1H), 5.13 (tt, J = 6.5, 3.3 Hz, 1H), 3.31 - 3.21 (m, 2H), 3.19 - 3.10 (m, 2H), 2.07 - 1.96 (m, 2H), 1.92 - 1.82 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.97, 162.43, 161.02, 158.93, 158.59, 158.25, 157.90, 156.33, 120.02, 118.67, 117.62, 73.34, 50.00, 43.41, 30.31. HRMS (ESI, m / z): [M+H] + calcd for C 10 H 12 NO4 + 210.0761; found 210.0769.

[0078] Example 10:

[0079] The method is the same as in Example 8, when R 3 represent Compound VI-3 was prepared.

[0080]

[0081] VI-3 NMR and Mass Spectrometry Characterization: 1 H NMR(500MHz,DMSO-d6)δ9.13(brs,1H),8.90(d,J=1.0Hz,1H),8.89(brs,1H),8.25(dd,J=6.0,1.1 Hz,1H),6.49(d,J=5.9Hz,1H),5.37(tt,J=7.0,5.3Hz,1H),4.38–4.34(m,2H),4.13–4.06(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.70,163.38,161.67,159.09,158.74,156.94,119.85,119.18,65.63,52.59.HRMS(ESI,m / z):[M+H] + calcd for C9H9NO4 + 196.0604; found 196.0606.

[0082] Example 11:

[0083] Same as in Example 4, when R 2 represent Compound V-8 was prepared.

[0084]

[0085] V-8 NMR and mass spectrometry characterization: 1 H NMR (400MHz, Chloroform-d) δ8.47(d,J=1.1Hz,1H),7.75(dd,J=5.9,1.1Hz,1H),6.48(d,J=5.9Hz,1H),4.47–4.41(m,2H),3.93–3.86(m,2H),3.02(brs,1H). 13 C NMR(126MHz,Chloroform-d)δ174.06,163.28,161.16,154.72,121.81,119.89,67.27,60.52.HRMS(ESI,m / z):[M+Na] + calcd for C8H8NaO5+ 207.0264; found 207.0261.

[0086] Example 12:

[0087] The procedure of Example 4 was followed when R 2 represents Compound V-9 was prepared.

[0088]

[0089] NMR and mass spectrometry characterization of V-9: 1 H NMR (500 MHz, Chloroform-d) δ 8.49 (d, J = 1.1 Hz, 1H), 7.72 (dd, J = 5.9, 1.1 Hz, 1H), 6.46 (d, J = 5.9 Hz, 1H), 4.48 - 4.42 (m, 2H), 3.85 - 3.80 (m, 2H), 3.78 - 3.73 (m, 2H), 3.68 - 3.63 (m, 2H), 2.66 (br s, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 173.90, 163.04, 161.57, 154.58, 121.42, 120.10, 72.48, 68.70, 64.59, 61.80. HRMS (ESI, m / z): [M + Na] + calcd for C 10 H 12 NaO6 + 251.0526; found 251.0522.

[0090] Example 13:

[0091] The procedure of Example 4 was followed when R 2 represents Compound V-10 was prepared.

[0092]

[0093] NMR and mass spectrometry characterization of V-10: 1H NMR (400 MHz, Chloroform-d) δ 8.57 (d, J = 1.1 Hz, 1H), 7.72 (dd, J = 5.9, 1.0 Hz, 1H), 6.88 (brs, 1H), 6.46 (d, J = 5.9, z 1H), 5.45 (t, J = 8.4 Hz, 1H), 3.55 - 3.35 (m, 2H), 2.77 - 2.67 (m, 1H), 2.28 - 2.13 (m, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 173.68, 168.98, 162.15, 161.09, 154.46, 121.64, 120.09, 70.44, 43.29, 38.38, 31.09, 30.21, 21.49. HRMS (ESI, m / z): [M + Na] + calcd for C 13 H 15 NNaO5 + 288.0842; found 288.0837.

[0094] Example 14:

[0095] The compound V-11 was prepared in the same manner as in Example 4, when R 2 represents The compound V-11 was prepared in the same manner as in Example 4, when R

[0096]

[0097] NMR and mass spectrum characterization of V-11: 1 H NMR (400 MHz, Chloroform-d) δ 8.57 (d, J = 1.1 Hz, 1H), 7.72 (dd, J = 5.9, 1.0 Hz, 1H), 6.88 (brs, 1H), 6.46 (d, J = 5.9, z 1H), 5.45 (t, J = 8.4 Hz, 1H), 3.55 - 3.35 (m, 2H), 2.77 - 2.67 (m, 1H), 2.28 - 2.13 (m, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 174.05, 173.02, 161.84, 161.58, 154.51, 120.06, 77.36, 70.95, 38.87, 28.23. HRMS (ESI, m / z): [M + Na] + calcd for C 10 H9NNaO5 +246.0373; found 246.0367.

[0098] Example 15:

[0099] The procedure of Example 4 was followed when R 2 represents Compound V-12 was prepared.

[0100]

[0101] NMR and mass spectrometry characterization of V-12: 1 H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 1.1 Hz, 1H), 7.73 (dd, J = 5.9, 1.1 Hz, 1H), 6.47 (d, J = 5.9 Hz, 1H), 6.03 (s, 1H), 5.64 - 5.55 (m, 1H), 3.85 (dd, J = 11.5, 6.0 Hz, 1H), 3.55 (ddd, J = 11.5, 2.3, 1.1 Hz, 1H), 2.80 (dd, J = 18.0, 7.2 Hz, 1H), 2.52 (dd, J = 17.9, 2.7 Hz, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 175.49, 173.61, 161.90, 161.57, 154.59, 120.61, 119.98, 70.66, 48.74, 36.94. HRMS (ESI, m / z): [M+Na] + calcd for C 10 H9NNaO5 + 246.0373; found 246.0369.

[0102] Example 16:

[0103] The procedure of Example 4 was followed when R 2 represents Compound V-13 was prepared.

[0104]

[0105] NMR and mass spectrometry characterization of V-13: 1H NMR (400 MHz, Chloroform-d) δ 8.41 (dd, J = 5.9, 1.1 Hz, 1H), 7.72 (ddd, J = 5.9, 4.7, 1.1 Hz, 1H), 6.44 (dd, J = 8.1, 5.8 Hz, 1H), 5.55 - 5.48 (m, 1H), 3.83 - 3.46 (m, 4H), 2.36 - 2.09 (m, 2H), 2.06 (d, J = 16.4 Hz, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 173.57, 169.59, 169.39, 162.24, 162.03, 161.21, 161.18, 154.53, 154.50, 121.35, 121.01, 120.09, 75.05, 73.84, 52.98, 51.43, 45.43, 43.68, 32.11, 30.38, 22.61, 22.45. HRMS (ESI, m / z): [M + Na] + calcd for C 12 H 13 NNaO5 + 274.0686; found 274.0681.

[0106] Example 17:

[0107] The compound V-14 was prepared according to the procedure of Example 4, when R 2 represents The compound V-14 was prepared according to the procedure of Example 4, when R

[0108]

[0109] NMR and mass spectrometry of V-14: 1 H NMR (400 MHz, Chloroform-d) δ 8.50 (d, J = 1.1 Hz, 1H), 7.74 (dd, J = 5.9, 1.1 Hz, 1H), 6.43 (d, J = 5.8 Hz, 1H), 5.32 (tt, J = 6.8, 4.2 Hz, 1H), 4.46 (ddd, J = 9.9, 6.8, 1.5 Hz, 1H), 4.32 (ddd, J = 11.3, 7.0, 1.4 Hz, 1H), 4.16 (ddd, J = 10.0, 4.2, 1.4 Hz, 1H), 4.02 (ddd, J = 11.2, 4.3, 1.4 Hz, 1H), 1.84 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 173.41, 170.58, 161.99, 161.87, 154.64, 120.28, 120.01, 63.74, 57.34, 54.62, 18.95. HRMS (ESI, m / z): [M+Na] + calcd for C 11 H 11 NNaO5 + 260.0529; found 260.0526.

[0110] Example 18:

[0111]

[0112] Compound 40 mg (0.17 mmol, 1.0 eq) was placed in a reaction bottle, 10 mL of dichloromethane was added to dissolve, and DMP 78 mg (0.18 mmol, 1.1 eq) was added. The reaction was carried out at 23 °C for 3 h. TLC monitoring showed that the reaction was complete, and the filtrate was extracted and dried on a column to obtain white solid 20 mg, with a yield of 50%

[0113] IX NMR and mass spectrometry characterization: 1 H NMR (500 MHz, Chloroform-d) δ 8.46 (d, J = 1.1 Hz, 1H), 7.75 (dd, J = 5.8, 1.1 Hz, 1H), 6.47 (d, J = 5.8 Hz, 1H), 4.82 (s, 2H), 4.19 (d, J = 2.4 Hz, 2H), 3.78 - 3.70 (m, 4H), 2.41 (t, J = 2.4 Hz, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 195.14, 175.50, 161.35, 154.89, 126.14, 120.14, 79.65, 77.63, 74.71, 70.73, 69.13, 58.44. HRMS (ESI, m / z): [M+Na]+ C 12 H 12 NaO5 + 259.0577; found 259.0572.

[0114] Example 19:

[0115]

[0116] Compound IX 23 mg (63.5 μmol, 1.0 eq), azidoacetic acid 10 mg (95.2 μmol, 1.5 eq) were added to a reaction vial, copper sulfate pentahydrate 2.4 mg (9.5 μmol, 0.15 eq), ascorbic acid 1.7 mg (9.5 μmol, 0.15 eq), t-butanol 0.5 mL, water 0.5 mL, stirred at 19 °C for 9 h. TLC monitored the reaction was complete, after dried, added 1 mL of acetonitrile / water (95 / 5, contain 0.1% trifluoroacetic acid), filtered, HPLC purification to get 13 mg oil, 61%.

[0117] X-1 NMR and mass spectrometry characterization: 1 H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 1.2 Hz, 1H), 7.83 (s, 1H), 7.76 (dd, J = 5.8, 1.2 Hz, 1H), 6.46 (d, J = 5.8 Hz, 1H), 5.12 (s, 2H), 4.80 (s, 2H), 4.68 (s, 2H), 3.72 (s, 4H). 13 C NMR (101 MHz, Chloroform-d, MeOD-d4) δ 195.20, 175.91, 168.13, 161.72, 155.22, 145.46, 125.92, 124.70, 119.95, 77.62, 70.80, 69.96, 64.56, 50.91. HRMS (ESI, m / z): [M-H] - C 14 H 14 N3NaO7 - 336.0837; found 236.08338.

[0118] Example 20:

[0119] The same method as Example 18, when R 5 represents Compound X-2 was prepared.

[0120]

[0121] X-2 NMR and mass spectrometry characterization: 1 H NMR (500 MHz, Chloroform-d, MeOD-d4) δ 8.52 (s, 1H), 8.25 (s, 1H), 7.79 (d, J = 4.9 Hz, 1H), 6.49 (d, J = 5.8 Hz, 1H), 5.01 (s, 2H), 4.81 (s, 2H), 4.64 (s, 2H), 4.16 (s, 2H), 3.72 (s, 4H), 3.18 (s, 9H). 13 CNMR (101 MHz, Chloroform-d, MeOD-d4) δ 195.14, 176.03, 161.92, 159.26, 155.54, 125.62, 124.53, 119.72, 77.36, 70.58, 69.96, 64.35, 64.19, 53.59, 44.09. HRMS (ESI, m / z): [M+Na] + calcd for C 17 H 25 N4O5 + 365.1819; found 365.1816.

[0122] Example 21:

[0123] IX Coupling to lysine residue

[0124]

[0125] Lysine derivative (N α -Cbz-Lys(NH2)-OH) was dissolved in pH 7.4 buffer (100 mM) to make a 4 mM stock solution, and the compound was dissolved in double distilled water to make a 4.8 mM stock solution, equal volume of the two solutions were mixed, and the reaction was carried out in a 37 °C water bath for 2 h. The system was diluted into acetonitrile / water (5:95, 0.1% trifluoroacetic acid) solution, purified by HPLC (gradient: 10% CH3CN 10 min, then 10% to 75% CH3CN 10 min, then 75% to 10% CH3CN 5 min, then 10% CH3CN 5 min) to obtain IX.

[0126] IX NMR and mass spectrometry characterization: 1H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 2.3 Hz, 1H), 7.41 - 7.27 (m, 5H), 6.67 (d, J = 7.5 Hz, 1H), 5.68 (d, J = 7.5 Hz, 1H), 5.08 (s, 2H), 4.89 (s, 2H), 4.36 (q, J = 6.4 Hz, 1H), 4.20 (d, J = 2.4 Hz, 4H), 3.89 (s, 2H), 3.75 (s, 4H), 2.43 (t, J = 2.4 Hz, 1H), 2.03 - 1.66 (m, 4H), 1.49 - 1.22 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 196.37, 176.99, 174.00, 156.10, 145.41, 140.19, 136.38, 128.70, 128.37, 128.22, 124.08, 122.88, 79.73, 77.99, 74.88, 70.53, 69.17, 67.11, 58.44, 57.59, 53.35, 31.58, 29.89, 21.32. HRMS (ESI, m / z): [M + Na] + calcd for C 22 H 31 N2O8 + 499.2075; found 499.2054.

[0127] Example 22:

[0128] Coupling of Compound IX to Polypeptide Sequence 1

[0129]

[0130] Polypeptide Sequence 1 was dissolved in pH 7.4 buffer (100 mM) to make a 2 mM stock solution, and Compound IX was dissolved in double distilled water to make a 6 mM stock solution. 50 μL of the polypeptide sequence 1 stock solution was taken in a 1.5 mL centrifuge tube, and 50 μL of the Compound IX stock solution was added. The reaction was carried out in a 37 °C water bath, and the coupling efficiency was monitored by HPLC. After 30 minutes, the coupling efficiency was about 100%. The specific reaction of Compound IX with lysine residues was verified by MS / MS (as shown in Figure 1

[0131] The same technical effects can be achieved by coupling the compounds of general formula (I) to polypeptide sequence 1, respectively.

[0132] Example 23:

[0133] Coupling of Compound IX to Polypeptide Sequence 2 ​

[0134]

[0135] The polypeptide sequence 2 was dissolved in a pH 7.4 buffer (100 mM) to prepare a 2 mM mother liquor, and the compound was dissolved in double-distilled water to prepare a 6 mM mother liquor. 50 μL of the polypeptide sequence 2 mother liquor was taken in a 1.5 ml centrifuge tube, 50 μL of the compound IX mother liquor was added, and the reaction was carried out in a 37 °C water bath. The coupling efficiency was monitored by HPLC. The reaction was carried out for 60 minutes, and the coupling efficiency was about 100%. The IX specifically reacted with the lysine residue, and the modification site was verified by secondary mass spectrometry (as shown in Figure 2 Figure 2

[0136] The polypeptide sequence 2 was dissolved in a pH 7.4 buffer (100 mM) to prepare a 2 mM mother liquor, and the compound was dissolved in double-distilled water to prepare a 6 mM mother liquor. 50 μL of the polypeptide sequence 2 mother liquor was taken in a 1.5 ml centrifuge tube, 50 μL of the compound IX mother liquor was added, and the reaction was carried out in a 37 °C water bath. The coupling efficiency was monitored by HPLC. The reaction was carried out for 60 minutes, and the coupling efficiency was about 100%. The IX specifically reacted with the lysine residue, and the modification site was verified by secondary mass spectrometry (as shown in

[0137] Example 24:

[0138] Coupling of IX to polypeptide sequence 3

[0139]

[0140] The polypeptide sequence 3 was dissolved in a pH 7.4 buffer (100 mM) to prepare a 2 mM mother liquor, and the compound was dissolved in double-distilled water to prepare a 6 mM mother liquor. 50 μL of the polypeptide sequence 3 mother liquor was taken in a 1.5 ml centrifuge tube, 50 μL of the compound IX mother liquor was added, and the reaction was carried out in a 37 °C water bath. The coupling efficiency was monitored by HPLC. The reaction was carried out for 60 minutes, and the coupling efficiency was about 100%. The IX specifically reacted with the lysine residue, and the modification site was verified by secondary mass spectrometry (as shown in Figure 3

[0141] The polypeptide sequence 3 was dissolved in a pH 7.4 buffer (100 mM) to prepare a 2 mM mother liquor, and the compound was dissolved in double-distilled water to prepare a 6 mM mother liquor. 50 μL of the polypeptide sequence 3 mother liquor was taken in a 1.5 ml centrifuge tube, 50 μL of the compound IX mother liquor was added, and the reaction was carried out in a 37 °C water bath. The coupling efficiency was monitored by HPLC. The reaction was carried out for 60 minutes, and the coupling efficiency was about 100%. The IX specifically reacted with the lysine residue, and the modification site was verified by secondary mass spectrometry (as shown in

[0142] Example 25:

[0143] Coupling of IX to lysozyme C

[0144]

[0145] wherein m represents the number of free amine groups in the protein, m = 1-7.

[0146] ​​​10 mg lysozyme C was dissolved in 20 ml pH 7.4 buffer (10 mM) to prepare a 34 μM solution, 7-112 equivalent compound IX was added to the lysozyme solution, which was shaken on a shaking table at 37 °C for 6 hours, then purified by dialysis with deionized water four times, and freeze-dried to obtain a white solid, and the molecular weight was identified by maldi-tof.

[0147] The same technical effects can be achieved by coupling the compound of general formula (I) with lysozyme C respectively.

[0148] It should be noted that the above examples are only preferred embodiments of the present application, and are not intended to limit the protection scope of the present application, and equivalent substitutions or replacements made on the basis of the above all belong to the protection scope of the present application.

Claims

1. A 3-acyl-4-pyranone derivative, characterized in that, Including compounds of general formula (I) or their chemically acceptable salts; The R is selected from:

2. The 3-acyl-4-pyranone derivative as described in claim 1, characterized in that, The chemically acceptable salt is an addition salt formed by the compound of general formula (I) and an acid selected from hydrogen chloride, hydrogen bromide, trifluoroacetic acid, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, acetic acid, or phosphoric acid.

3. The method for preparing the 3-acyl-4-pyranone derivative as described in claim 1, characterized in that, The synthesis route can be selected from any one of route 1, route 2, or route 3; Route 1: Where R 1 represent Route 2: Where R 2 represent R 3 represent Route 3: Where R 4 represent R 5 represent 4. The preparation method according to claim 3, characterized in that, The specific steps of Route 1 are as follows: using compound II as the initial raw material, it is acid-catalyzed hydrolyzed to obtain compound III. After compound III reacts with oxalyl chloride or thionyl chloride, it undergoes an acylation reaction with an amine to obtain compound IV. The specific steps of Route 2 are as follows: using compound II as the initial raw material, it is hydrolyzed by acid catalysis to obtain compound III, which is then esterified with an alcohol to obtain compound V, and deprotected by acid catalysis to obtain VI. The specific steps of Route 3 are as follows: using compound VII as the initial raw material, reacting it with aldehyde under alkaline conditions to obtain VIII, obtaining compound IX under oxidizing conditions, and obtaining X through a click chemical reaction.

5. The use of the 3-acyl-4-pyranone derivative as described in any one of claims 1-4 in the preparation of peptide and protein coupling reagents.

6. The application as described in claim 5, characterized in that, The method for coupling the 3-acyl-4-pyranone derivative compound with a polypeptide or protein is as follows: the 3-acyl-4-pyranone derivative reacts with the polypeptide or protein in a reaction solvent at a pH of 6.5-10.0 and a reaction temperature of 0-40°C to obtain the coupling product; the reaction solvent is selected from any one or a combination of several of water, pH buffer, acetonitrile, dimethyl sulfoxide, and N,N-dimethylformamide.

7. A class of polypeptide conjugates, characterized in that, It is formed by coupling the compound of formula (I) as described in claim 1 with a polypeptide, and has the structure shown in formula (XI):

8. A class of protein-coupled products, characterized in that, It is formed by coupling the compound of formula (I) as described in claim 1 with a protein, and has the structure shown in formula (XII): Where m represents the number of free amino groups in the protein, m = 1 to 7.

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