CN117050046A - 3-acyl-4-pyrone derivative as coupling reagent and application thereof in preparation of polypeptide and protein conjugate - Google Patents

3-acyl-4-pyrone derivative as coupling reagent and application thereof in preparation of polypeptide and protein conjugate Download PDF

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CN117050046A
CN117050046A CN202310848377.1A CN202310848377A CN117050046A CN 117050046 A CN117050046 A CN 117050046A CN 202310848377 A CN202310848377 A CN 202310848377A CN 117050046 A CN117050046 A CN 117050046A
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姚祝军
农克意
赵益禄
奚婕
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Nanjing University
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    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
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    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
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Abstract

The invention belongs to the technical field of organic synthesis and protein modification, and particularly relates to a 3-acyl-4-pyrone derivative which is used as a coupling reagent and application thereof in preparation of polypeptide and protein conjugates, wherein the 3-acyl-4-pyrone derivative can be quickly and efficiently subjected to O-N insertion substitution reaction with lysine residues in polypeptide or protein sequences in a chemoselective manner under a near physiological condition so as to realize coupling of the polypeptide and the protein; the synthesis method of the 3-acyl-4-pyrone derivative is simple, the modification operation on the protein is simple and convenient, the structure is novel, no relevant report on application to polypeptide and protein modification exists yet, and a novel technology and a novel mode are provided for various functional modification of the polypeptide and the protein.

Description

3-acyl-4-pyrone derivative as coupling reagent and application thereof in preparation of polypeptide and protein conjugate
Technical Field
The invention 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
Chemical modification of polypeptides or proteins is an important method for improving physicochemical properties, pharmacokinetic properties, and biological activities, such as increasing stability, prolonging the duration of action in vivo, increasing therapeutic effects, and reducing toxic and side effects. By introducing specific functional groups such as guide groups, fluorophores, drugs and the like, the distribution of polypeptides or proteins in a life system can be changed, biomedical imaging can be performed, disease treatment can be performed and the like. In general, coupling reagents need to react with some of the functional groups in polypeptides or proteins, lysine being an important modification site due to its high content in proteins, which is about 5.9% of the human proteome, and typically exposed on the surface of proteins.
Classical modification of lysine residues in polypeptides or proteins is the formation of stable amide linkages with lysine residues using pre-prepared N-hydroxysuccinimide (NHS) esters. However, NHS esters are easily hydrolyzed in aqueous solutions and react with tyrosine, serine and threonine to form byproducts, and chemical modification often results in reduced protein activity, limiting their use. Therefore, development of a novel lysine coupling method for realizing efficient modification of polypeptides and proteins is needed.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides a polypeptide and protein coupling reagent containing a 3-substituted-4-pyrone parent nucleus structure. The structural parent nucleus is simplified from a natural product azafedone, and can be quickly and efficiently subjected to O-N insertion substitution reaction with lysine residues in a polypeptide or protein sequence in a chemical selectivity manner under a near physiological condition, so that the coupling of the polypeptide and the protein is realized.
The technical scheme of the invention is as follows:
a 3-acyl-4-pyrone derivative comprising a compound of formula (I) or a chemically acceptable salt thereof;
wherein R is selected from carboxyl, amino, hydroxyl, alkynyl, azido, sulfhydryl, succinimide or maleimide.
Preferably, said R is selected from
Preferably, the chemically acceptable salt is an addition salt of the compound of formula (I) with an acid selected from hydrogen chloride, hydrogen bromide, trifluoroacetic acid, sulfuric acid, p-toluene sulfonic acid, benzenesulfonic acid, methanesulfonic acid, acetic acid or phosphoric acid.
Preferably, the R group serves as a derivatization site for introducing a modifying group. More preferably, the modifying group is selected from fluorophores, chemical and biological molecules with biological functions, or modifying groups.
The first preparation method of the 3-acyl-4-pyrone derivative comprises the following synthetic route:
wherein R is 1 Representative of
Preferably, the preparation method comprises the following steps: the compound II is used as an initial raw material, and is subjected to acid catalytic hydrolysis to obtain a compound III, and the compound III reacts with oxalyl chloride and thionyl chloride to generate an acylation reaction with amine to obtain a compound IV.
Preferably, the preparation method comprises the following steps: dissolving the compound II in dichloromethane, dropwise adding trifluoroacetic acid and triethylsilane at the temperature of minus 15 ℃ under the protection of nitrogen; spin-drying the solution after the reaction to obtain a white solid; adding dichloromethane to the white solid at 0 ℃ under nitrogen protection, dissolving, then dropwise adding oxalyl chloride and DMF, and stirring for 2h at 0 ℃; spin-drying after the reaction is finished, adding dichloromethane for dissolution, and stirring at 0 ℃; slowly dripping dichloromethane solution of ethanolamine, maintaining the temperature and stirring to obtain the product.
A second preparation method of the 3-acyl-4-pyrone derivative comprises the following synthetic route:
wherein R is 2 Representative of
R 3 Representative of
Preferably, the preparation method comprises the following steps: the method comprises the steps of taking a compound II as an initial raw material, carrying out acid catalytic hydrolysis to obtain a compound III, carrying out esterification reaction on the compound III and alcohol to obtain a compound V, and carrying out acid catalytic deprotection to obtain a VI.
Preferably, the preparation method comprises the following steps: dissolving the compound II in 4.0mL of dichloromethane, and dropwise adding trifluoroacetic acid and triethylsilane at the temperature of minus 15 ℃ under the protection of nitrogen; spin drying after the reaction is finished to obtain white solid; adding dichloromethane into the white solid and alcohol under the protection of nitrogen to dissolve, and stirring at 0 ℃; EDCI and DMAP are dissolved in dichloromethane and added into a reaction system in a dropwise manner, and the mixture is reacted for 18 hours at 24 ℃ to obtain a product.
A third preparation method of the 3-acyl-4-pyrone derivative is as follows:
wherein R is 4 Representative ofR 5 Represents->
Preferably, the preparation method comprises the following steps: the method comprises the steps of taking a compound VII as an initial raw material, reacting with aldehyde under the action of alkali to obtain VIII, reacting with an oxidant to obtain a compound IX, and performing click chemistry reaction to obtain X.
Preferably, the preparation method comprises the following steps: adding anhydrous methanol to dissolve the compound VII under the protection of nitrogen, and stirring at 0 ℃; dissolving sodium methoxide in methanol, adding the methanol into a reaction system, then adding aldehyde, and reacting for 10 hours at the temperature of 23 ℃; adding glacial acetic acid for quenching after the reaction is finished, stirring and spin-drying to obtain gray solid; the grey solid was dissolved in dichloromethane and DMP was added and reacted at 23 ℃ for 3h to give the product.
The 3-acyl-4-pyrone derivatives are useful as polypeptide and protein coupling reagents. The coupling reagent can react with lysine in polypeptide or protein sequence fast and effectively to realize coupling of polypeptide and protein.
A method for coupling a polypeptide or protein from said 3-acyl-4-pyrone derivative comprising the steps of: in water, pH buffer solution, acetonitrile, dimethyl sulfoxide, N-dimethylformamide or a mixture thereof, the reaction pH is 6.5-10.0, and the compound of the formula (I) is coupled with primary amine in polypeptide or protein at the reaction temperature of 0-40 ℃ to obtain a polypeptide coupling product shown as the formula (XI) or a protein coupling product shown as the formula (XII).
Wherein m represents the number of free amine groups in the protein, and m=1 to 7.
Preferably, the polypeptide is polypeptide sequence 1 (AcNH-DRVYIHGKF-CONH 2 ) Polypeptide sequence 2 (AcNH-QAWECMKF-CONH 2 ) Or polypeptide sequence 3 (AcNH-APSKF-CONH) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The protein is Lysozyme (Lysozyme C)。
The advantages of the present invention over the prior art are as follows,
the invention simplifies the structure of the natural product azafedone, and the obtained polypeptide and protein coupling reagent containing 3-substituted-4-pyrone structure has novel structure, high solubility, simple and convenient modification operation on protein and rapid reaction, has not been applied to polypeptide and protein modification in a related report, and provides a new technology and a new mode for various functional modification of polypeptide and protein.
Drawings
FIG. 1 is a secondary mass spectrum of the polypeptide conjugate product P-1;
FIG. 2 is a secondary mass spectrum of the polypeptide conjugate product P-2;
FIG. 3 is a secondary mass spectrum of the polypeptide conjugate product P-3;
FIG. 4 is a mass spectrum (maldi-tof) of lysozyme (lysozyme C) conjugate product.
Detailed Description
Compound II was synthesized by the method reported in patent (CN 113444102A).
Example 1:
compound II 50mg (0.25 mmol,1.0 eq) was dissolved in 4.0mL of dichloromethane and trifluoroacetic acid 0.38mL (5.1 mmol,20 eq) and triethylsilane 0.41mL (2.5 mmol,10 eq) were added dropwise at-15℃under nitrogen. After the completion of the reaction, TLC was monitored, the solution was dried by spinning to give 35mg of a white solid. 100mg (0.71 mmol,1.0 eq) of white solid was added to the reaction flask, dissolved in 30mL of dichloromethane under nitrogen at 0deg.C, followed by dropwise addition of 135mg (0.11 mmol,1.5 eq) of oxalyl chloride, one drop of DMF, and stirring at 0deg.C for 2h. After the completion of the reaction, TLC was followed by spin-drying, and 5mL of methylene chloride was added for dissolution, followed by stirring at 0 ℃. A solution of 65mg of ethanolamine (0.10 mmol,1.4 eq) in dichloromethane was slowly added dropwise, and the temperature was maintained under stirring. After the completion of the reaction, the reaction was monitored by TLC and dried by spin-drying, followed by column chromatography on silica gel (dichloromethane: methanol=80:1) to give 42mg,32% of an oil.
IV-1 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ9.53(s,1H),8.76(d,J＝1.1Hz,1H),7.82(dd,J＝5.7,1.1Hz,1H),6.50(d,J＝5.7Hz,1H),3.76(s,2H),3.56(q,J＝5.4Hz,2H),3.30(brs,1H). 13 C NMR(126MHz,Chloroform-d)δ177.74,163.49,161.89,155.63,120.66,118.93,62.28,42.55.HRMS(ESI,m/z):[M+Na] + calcd for C 8 H 9 NNaO 4 + 184.0604；found 184.0603.
example 2:
in the same manner as in example 1, when R 1 Representative ofThe compound IV-2 is prepared.
IV-2 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d)δ9.69(brs,1H),8.78(d,J＝1.0Hz,1H),7.83(dd,J＝5.8,1.1Hz,1H),6.54(d,J＝5.7Hz,1H),4.24(q,J＝7.1Hz,2H),4.18(d,J＝5.6Hz,2H),1.30(t,J＝7.1Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ177.60,169.34,162.66,161.93,155.55,120.57,119.11,61.57,41.45,14.28.HRMS(ESI,m/z):[M+Na] + calcd for C 10 H 11 NNaO 5 + 248.0529；found 248.0526.
example 3:
in the same manner as in example 1, when R 1 Representative ofThe compound IV-3 is prepared.
IV-3 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ9.49(s,1H),8.80(d,J＝1.1Hz,1H),7.83(dd,J＝5.7,1.1Hz,1H),6.54(d,J＝5.7Hz,1H),4.20(dd,J＝5.5,2.6Hz,2H),2.24(t,J＝2.5Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ177.59,162.15,161.99,155.60,120.61,119.09,79.34,71.51,28.93.HRMS(ESI,m/z):[M+Na] + calcd for C 9 H 7 NNaO 3 + 178.0499；found 178.0498.
example 4:
compound 7.50 mg (0.25 mmol,1.0 eq) are dissolved in 4.0mL dichloromethane and trifluoroacetic acid 0.38mL (5.1 mmol,20 eq) and triethylsilane 0.41mL (2.5 mmol,10 eq) are added dropwise at-15℃under nitrogen. After the completion of the reaction, TLC was monitored, the solution was dried by spinning to give 35mg of a white solid. 49mg (0.35 mmol,2.0 eq) of white solid, 43mg (0.18 mmol,1.0 eq) of alcohol are added to the reaction flask, dissolved in 5mL of dichloromethane under nitrogen and stirred at 0deg.C. EDCI 67mg (0.35 mmol,2.0 eq), DMAP 1mg (0.008 mmol,0.1 eq) was dissolved in dichloromethane 1mL, added dropwise to the reaction system, and reacted at 24℃for 18h. After the completion of the reaction, TLC was followed by dilution with methylene chloride, washing with saturated sodium chloride once, and drying over anhydrous sodium sulfate. Silica gel column chromatography (dichloromethane: methanol=60:1) gave 24mg of oil in 29% yield
V-1 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.53(d,J＝1.1Hz,1H),7.76(dd,J＝5.9,1.1Hz,1H),6.48(d,J＝5.9Hz,1H),5.40(tt,J＝6.8,4.3Hz,1H),4.72(ddd,J＝11.0,6.7,1.5Hz,1H),4.42(dddd,J＝16.9,11.1,5.7,1.5Hz,2H),4.08-4.15(m,3H),3.86–3.61(m,6H),3.40(dd,J＝5.6,4.4Hz,2H). 13 C NMR(101MHz,Chloroform-d)δ173.37,169.35,161.96,161.78,154.49,120.36,120.04,70.86,70.76,70.49,70.04,65.27,58.32,55.18,50.71.HRMS(ESI,m/z):[M+Na] + calcd for C 15 H 18 N 4 NaO 7 + 389.1068；found 389.1063.
example 5:
as in example 4, when R 2 Represents R 2 Representative ofTo obtain the compound V-2.
V-2 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.42(d,J＝1.1Hz,1H),7.70(dd,J＝5.9,1.1Hz,1H),6.44(d,J＝5.9Hz,1H),5.00(p,J＝6.2Hz,1H),1.68(qd,J＝7.5,6.0Hz,4H),0.94(t,J＝7.5Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ173.94,162.36,160.53,154.37,121.90,119.96,78.25,26.43,9.67.HRMS(ESI,m/z):[M+Na] + calcd for C 11 H 14 NaO 4 + 233.0784；found 233.0780.
example 6:
as in example 4, when R 2 Representative ofTo obtain the compound V-3.
V-3 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d)δ8.41(s,1H),7.70(d,J＝5.9Hz,1H),6.43(d,J＝5.7Hz,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(126MHz,Chloroform-d)δ173.98,161.94,160.73,154.33,122.02,120.04,73.99,31.57,25.46,23.69.HRMS(ESI,m/z):[M+Na] + calcd for C 12 H 14 NaO 4 + 245.0784；found 235.0785.
example 7:
as in example 4, when R 2 Representative ofTo obtain the compound IV-3
V-4 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.38(d,J＝1.0Hz,1H),7.69(dd,J＝5.9,1.1Hz,1H),6.43(d,J＝5.9Hz,1H),5.36(tt,J＝6.2,2.7Hz,1H),1.99–1.86(m,2H),1.86–1.69(m,4H),1.69–1.56(m,2H). 13 C NMR(101MHz,Chloroform-d)δ173.95,162.34,160.70,154.35,121.95,120.02,78.56,32.77,23.84.HRMS(ESI,m/z):[M+Na] + calcd for C 11 H 12 NaO 4 + 231.0628；found 231.0623.
example 8:
as in example 4, when R 3 Representative ofTo obtain the compound V-5. The compound was dissolved in trifluoroacetic acid, dichloromethane=1:10, stirred at 0 ℃ for 3h and dried by spin to give a red oil vi-1.
VI-1 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,DMSO-d 6 )δ8.86(s,1H),8.63(s,2H),8.23(d,J＝5.5Hz,1H),6.46(d,J＝5.8Hz,1H),5.13(tt,J＝6.5,3.3Hz,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(126MHz,DMSO-d 6 )δ173.60,162.45,162.00,158.87,158.60,156.85,121.20,119.19,67.23,40.44,27.19.HRMS(ESI,m/z):[M+H] + calcd for C 11 H 14 NO 4 + 224.0917；found 224.0915.
example 9:
in the same manner as in example 8, when R 3 Representative ofTo obtain the compound VI-2.
VI-2 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,DMSO-d 6 )δ9.16(d,J＝37.8Hz,2H),8.78(s,1H),8.14(d,J＝6.0Hz,1H),6.38(d,J＝5.9Hz,1H),5.38(td,J＝4.4,2.0Hz,1H),3.44–3.15(m,4H),2.20–1.97(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ172.97,162.43,161.02,158.93,158.59,158.25,157.90,156.33,120.02,118.67,117.62,73.34,50.00,43.41,30.31.HRMS(ESI,m/z):[M+H] + calcd for C 10 H 12 NO 4 + 210.0761；found 210.0769.
example 10:
in the same manner as in example 8, when R 3 Representative ofTo obtain the compound VI-3.
VI-3 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,DMSO-d 6 )δ9.13(brs,1H),8.90(d,J＝1.0Hz,1H),8.89(brs,1H),8.25(dd,J＝6.0,1.1Hz,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-d 6 )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 C 9 H 9 NO 4 + 196.0604；found 196.0606.
example 11:
as in example 4, when R 2 Representative ofTo obtain the compound V-8.
V-8 nuclear magnetism and mass spectrum 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 C 8 H 8 NaO 5 + 207.0264；found 207.0261.
example 12:
as in example 4, when R 2 Representative ofTo obtain the compound V-9.
V-9 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d)δ8.49(d,J＝1.1Hz,1H),7.72(dd,J＝5.9,1.1Hz,1H),6.46(d,J＝5.9Hz,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(brs,1H). 13 C NMR(126MHz,Chloroform-d)δ173.90,163.04,161.57,154.58,121.42,120.10,72.48,68.70,64.59,61.80.HRMS(ESI,m/z):[M+Na] + calcd for C 10 H 12 NaO 6 + 251.0526；found 251.0522.
example 13:
as in example 4, when R 2 Representative ofTo obtain the compound V-10.
V-10 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d)δ8.43(s,1H),7.72(dd,J＝5.8,1.1Hz,1H),6.46–6.40(m,1H),5.25–5.18(m,1H),3.83–3.74(m,1H),3.74–3.65(m,1H),3.65–3.56(m,1H),3.46–3.37(m,1H),2.09(s,3H),1.98–1.86(m,2H),1.85–1.73(m,2H). 13 C NMR(101MHz,Chloroform-d)δ173.68,168.98,162.15,161.09,154.46,121.64,120.09,70.44,43.29,38.38,31.09,30.21,21.49.HRMS(ESI,m/z):[M+Na] + calcd for C 13 H 15 NNaO 5 + 288.0842；found 288.0837.
example 14:
as in example 4, when R 2 Representative ofTo obtain the compound V-11.
V-11 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.57(d,J＝1.1Hz,1H),7.72(dd,J＝5.9,1.0Hz,1H),6.88(brs,1H),6.46(d,J＝5.9,z1H),5.45(t,J＝8.4Hz,1H),3.55–3.35(m,2H),2.77–2.67(m,1H),2.28–2.13(m,1H). 13 C NMR(101MHz,Chloroform-d)δ174.05,173.02,161.84,161.58,154.51,120.06,77.36,70.95,38.87,28.23.HRMS(ESI,m/z):[M+Na] + calcd for C 10 H 9 NNaO 5 + 246.0373；found 246.0367.
example 15:
as in example 4, when R 2 Representative ofTo obtain the compound V-12.
V-12 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.47(d,J＝1.1Hz,1H),7.73(dd,J＝5.9,1.1Hz,1H),6.47(d,J＝5.9Hz,1H),6.03(s,1H),5.64–5.55(m,1H),3.85(dd,J＝11.5,6.0Hz,1H),3.55(ddd,J＝11.5,2.3,1.1Hz,1H),2.80(dd,J＝18.0,7.2Hz,1H),2.52(dd,J＝17.9,2.7Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ175.49,173.61,161.90,161.57,154.59,120.61,119.98,70.66,48.74,36.94.HRMS(ESI,m/z):[M+Na] + calcd for C 10 H 9 NNaO 5 + 246.0373；found 246.0369.
example 16:
as in example 4, when R 2 Representative ofTo obtain the compound V-13.
V-13 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.41(dd,J＝5.9,1.1Hz,1H),7.72(ddd,J＝5.9,4.7,1.1Hz,1H),6.44(dd,J＝8.1,5.8Hz,1H),5.55–5.48(m,1H),3.83–3.46(m,4H),2.36–2.09(m,2H),2.06(d,J＝16.4Hz,3H). 13 C NMR(126MHz,Chloroform-d)δ173.57,169.59,169.39,162.24,162.03,161.21,161.18,154.53,154.50,121.35,121.01,120.09,75.05,73.84,52.98,51.43,45.43,43.68,32.11,30.38,22.61,22.45.HRMS(ESI,m/z):[M+Na] + calcd for C 12 H 13 NNaO 5 + 274.0686；found 274.0681.
example 17:
as in example 4, when R 2 Representative ofTo obtain the compound V-14.
V-14 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.50(d,J＝1.1Hz,1H),7.74(dd,J＝5.9,1.1Hz,1H),6.43(d,J＝5.8Hz,1H),5.32(tt,J＝6.8,4.2Hz,1H),4.46(ddd,J＝9.9,6.8,1.5Hz,1H),4.32(ddd,J＝11.3,7.0,1.4Hz,1H),4.16(ddd,J＝10.0,4.2,1.4Hz,1H),4.02(ddd,J＝11.2,4.3,1.4Hz,1H),1.84(s,3H). 13 C NMR(101MHz,Chloroform-d)δ173.41,170.58,161.99,161.87,154.64,120.28,120.01,63.74,57.34,54.62,18.95.HRMS(ESI,m/z):[M+Na] + calcd for C 11 H 11 NNaO 5 + 260.0529；found 260.0526.
example 18:
/>
132mg (1.38 mmol,1.4 eq) of gamma-pyrone was added to the reaction flask, and 2mL of anhydrous methanol was added thereto under nitrogen protection, followed by stirring at 0 ℃. 26mg (0.49 mmol,0.5 eq) of sodium methoxide was dissolved in 1mL of methanol, and then 140mg (0.98 mmol,1.0 eq) of aldehyde was added to the reaction system, followed by reaction at 23℃for 10 hours. After the TLC monitoring reaction, adding glacial acetic acid for quenching, stirring for 15min, spin-drying, adding dichloromethane for dissolving, washing once with saturated sodium chloride, and drying with anhydrous sodium sulfate. Silica gel column chromatography (dichloromethane: acetone=40:1) gave 105mg of grey solid in 45% yield. 40mg (0.17 mmol,1.0 eq) of the compound was placed in a reaction flask, 10mL of methylene chloride was added for dissolution, 78mg (0.18 mmol,1.1 eq) of DMP was added, and the mixture was reacted at 23℃for 3 hours. After the TLC monitoring reaction is finished, suction filtration is carried out, and the filtrate is subjected to spin-dry column chromatography to obtain white solid 20mg with the yield of 50%
IX nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d)δ8.46(d,J＝1.1Hz,1H),7.75(dd,J＝5.8,1.1Hz,1H),6.47(d,J＝5.8Hz,1H),4.82(s,2H),4.19(d,J＝2.4Hz,2H),3.78–3.70(m,4H),2.41(t,J＝2.4Hz,1H). 13 C NMR(126MHz,Chloroform-d)δ195.14,175.50,161.35,154.89,126.14,120.14,79.65,77.63,74.71,70.73,69.13,58.44.HRMS(ESI,m/z):[M+Na] + calcd for C 12 H 12 NaO 5 + 259.0577；found 259.0572.
example 19:
23mg (63.5. Mu. Mol,1.0 eq) of Compound IX, 10mg (95.2. Mu. Mol,1.5 eq) of azidoacetic acid, 2.4mg (9.5. Mu. Mol,0.15 eq) of copper sulfate pentahydrate, 1.7mg (9.5. Mu. Mol,0.15 eq) of ascorbic acid, 0.5mL of t-butanol, 0.5mL of water and stirring at 19℃for 9h were added to a reaction flask. After TLC monitoring the reaction was complete, 1mL of acetonitrile/water (95/5, 0.1% trifluoroacetic acid) was added after pumping, and after filtration, HPLC purification gave 13mg of oil, 61%.
X-1 nuclear magnetism and mass spectrum characterization: 1 H NMR(400MHz,Chloroform-d)δ8.47(d,J＝1.2Hz,1H),7.83(s,1H),7.76(dd,J＝5.8,1.2Hz,1H),6.46(d,J＝5.8Hz,1H),5.12(s,2H),4.80(s,2H),4.68(s,2H),3.72(s,4H). 13 C NMR(101MHz,Chloroform-d，MeOD-d 4 )δ195.20,175.91,168.13,161.72,155.22,145.46,125.92,124.70,119.95,77.62,70.80,69.96,64.56,50.91.HRMS(ESI,m/z):[M-H] - calcd for C 14 H 14 N 3 NaO 7 - 336.0837；found 236.08338.
example 20:
in the same manner as in example 18, when R 5 Representative ofThe compound X-2 is obtained.
X-2 nuclear magnetism and mass spectrum characterization: 1 H NMR(500MHz,Chloroform-d，MeOD-d 4 )δ8.52(s,1H),8.25(s,1H),7.79(d,J＝4.9Hz,1H),6.49(d,J＝5.8Hz,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(101MHz,Chloroform-d，MeOD-d 4 )δ195.14,176.03,161.92,159.26,155.54,125.62,124.53,119.72,77.36,70.58,69.96,64.35,64.19,53.59,44.09.HRMS(ESI,m/z):[M+Na] + calcd for C 17 H 25 N 4 O 5 + 365.1819；found365.1816.
example 21:
coupling of IX to lysine residues
Lysine derivative (N) α -Cbz-Lys(NH 2 ) -OH) was dissolved in pH 7.4 buffer (100 mM) to prepare a 4mM stock solution, the compound was dissolved in secondary water to prepare a 4.8mM stock solution, and an equal volume of the solution was mixed and reacted in a 37 ℃ water bath for 2 hours. The system was diluted to acetonitrile/water (5:95, 0.1% trifluoroacetic acid) solution and purified by HPLC (gradient: 10% CH 3 CN 10min, followed by 10% to 75% CH 3 CN 10min, 75% to 10% CH 3 CN 5min, 10% CH then 3 CN 5 min), to obtain XI.
XI Nuclear magnetism and Mass Spectrometry characterization: 1 H NMR(400MHz,Chloroform-d)δ8.26(d,J＝2.3Hz,1H),7.41–7.27(m,5H),6.67(d,J＝7.5Hz,1H),5.68(d,J＝7.5Hz,1H),5.08(s,2H),4.89(s,2H),4.36(q,J＝6.4Hz,1H),4.20(d,J＝2.4Hz,4H),3.89(s,2H),3.75(s,4H),2.43(t,J＝2.4Hz,1H),2.03–1.66(m,4H),1.49–1.22(m,2H). 13 C NMR(101MHz,Chloroform-d)δ196.37,176.99,174.00,156.10,145.41,140.19,136.38,128.70,128.37,128.22,124.08,122.88,79.73,77.99,74.88,70.53,69.17,67.11,58.44,57.59,53.35,31.58,29.89,21.32.HRMS(ESI,m/z):[M+Na] + calcd for C 22 H 31 N 2 O 8 + 499.2075；found 499.2054.
example 22:
conjugation of IX to polypeptide sequence 1
The polypeptide sequence 1 was dissolved in pH 7.4 buffer (100 mM) to prepare a 2mM stock solution, the compound was dissolved in secondary water to prepare a 6mM stock solution, 50. Mu.L of the polypeptide sequence 1 stock solution was taken in a 1.5ml centrifuge tube, 50. Mu.L of the compound IX stock solution was added to perform a reaction in a 37℃water bath, and the coupling efficiency was monitored by HPLC for 30 minutes and was about 100%. IX specificity reacted with lysine residues and the modification site was verified by secondary mass spectrometry (as shown in FIG. 1).
The same technical effects can be obtained by coupling the compounds of the general formula (I) with the polypeptide sequence 1 respectively.
Example 23:
conjugation of IX to polypeptide sequence 2
The polypeptide sequence 2 was dissolved in pH 7.4 buffer (100 mM) to prepare a 2mM stock solution, the compound was dissolved in secondary water to prepare a 6mM stock solution, 50. Mu.L of the polypeptide sequence 2 stock solution was taken in a 1.5ml centrifuge tube, 50. Mu.L of the compound IX stock solution was added to perform a reaction in a 37℃water bath, and the coupling efficiency was monitored by HPLC for 60 minutes and was about 100%. IX specificity reacted with lysine residues and the modification site was verified by secondary mass spectrometry (as shown in FIG. 2). IX specificity reacted with lysine residues and the modification site was verified by secondary mass spectrometry (as shown in FIG. 2).
The same technical effects can be obtained by coupling the compounds of the general formula (I) with the polypeptide sequence 2 respectively.
Example 24:
conjugation of IX to polypeptide sequence 3
The polypeptide sequence 3 was dissolved in pH 7.4 buffer (100 mM) to prepare a 2mM stock solution, the compound was dissolved in secondary water to prepare a 6mM stock solution, 50. Mu.L of the polypeptide sequence 3 stock solution was taken in a 1.5ml centrifuge tube, 50. Mu.L of the compound IX stock solution was added to perform a reaction in a 37℃water bath, and the coupling efficiency was monitored by HPLC for 15 minutes and was about 100%. IX specificity reacted with lysine residues and the modification site was verified by secondary mass spectrometry (as shown in FIG. 3).
The same technical effects can be obtained by coupling the compounds of the general formula (I) with the polypeptide sequence 3 respectively.
Example 25:
coupling of IX to lysozyme (lysozyme C)
Wherein m represents the number of free amine groups in the protein, and m=1 to 7.
10mg of lysozyme (lysozyme C) was dissolved in 20 ml of pH 7.4 buffer (10 mM) to prepare a 34. Mu.M solution, 7-112 equivalents of Compound IX was added to the lysozyme solution, and the mixture was subjected to shaking reaction at 37℃for 6 hours, followed by dialysis against deionized water for purification four times, and freeze-dried to obtain a white solid, which was identified as a molecular weight by maldi-tof.
The same technical effects can be obtained by coupling the compounds of the general formula (I) with lysozyme (lysozyme C), respectively.
It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and the equivalents or alternatives made on the basis of the above description are all included in the scope of the present invention.

Claims (10)

1. A 3-acyl-4-pyrone derivative comprising a compound of formula (I) or a chemically acceptable salt thereof;
the R is selected from carboxyl, amino, hydroxyl, alkynyl, azido, sulfhydryl, succinimide or maleimide.
2. The 3-acyl-4-pyrone derivative according to claim 1, wherein R is selected from the group consisting of:
3. the 3-acyl-4-pyrone derivative according to claim 1 wherein the chemically acceptable salt is an addition salt of the compound of formula (I) with an acid selected from hydrogen chloride, hydrogen bromide, trifluoroacetic acid, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, acetic acid or phosphoric acid.
4. The 3-acyl-4-pyrone derivative according to claim 1 wherein the R group is introduced as a derivatization site with a modifying group; the modifying group is selected from a fluorophore, a chemical and biological molecule with biological function or a modifying group.
5. The process for the preparation of 3-acyl-4-pyrone derivatives according to claim 1, wherein the synthetic route is selected from any one of route 1, route 2 and route 3;
route 1:
wherein R is 1 Representative of
Route 2:
wherein R is 2 Representative of
R 3 Representative of
Route 3:
wherein R is 4 Representative ofR 5 Represents->
6. The method according to claim 5, wherein,
the specific steps of the route 1 are as follows: the method comprises the steps of taking a compound II as an initial raw material, carrying out acid catalytic hydrolysis to obtain a compound III, reacting the compound III with oxalyl chloride and thionyl chloride, and carrying out acylation reaction with amine to obtain a compound IV;
the specific steps of the route 2 are as follows: taking a compound II as an initial raw material, carrying out acid catalytic hydrolysis to obtain a compound III, carrying out esterification reaction on the compound III and alcohol to obtain a compound V, and carrying out acid catalytic deprotection to obtain a VI;
the specific steps of the route 3 are as follows: the method comprises the steps of taking a compound VII as an initial raw material, reacting with aldehyde under the action of alkali to obtain VIII, reacting with an oxidant to obtain a compound IX, and performing click chemistry reaction to obtain X.
7. Use of a 3-acyl-4-pyrone derivative according to any one of claims 1 to 6 in polypeptide and protein coupling reagents.
8. The use according to claim 7, wherein the 3-acyl-4-pyrone derivative compound is used for coupling a polypeptide or protein by: the 3-acyl-4-pyrone derivative reacts with polypeptide or protein in a reaction solvent at the pH of 6.5-10.0 and the reaction temperature of 0-40 ℃ to prepare a coupling product; the reaction solvent is selected from any one or a combination of a plurality of water, pH buffer solution, acetonitrile, dimethyl sulfoxide and N, N-dimethylformamide.
9. A polypeptide coupling product, which is formed by coupling a compound of formula (I) as defined in claim 1 with a polypeptide, and has a structure as defined in formula (xi):
10. a protein coupling product, which is characterized by being formed by coupling a compound of formula (I) as defined in claim 1 with a protein, and having a structure as shown in formula (xii):
wherein m represents the number of free amine groups in the protein, and m=1 to 7.
CN202310848377.1A 2023-07-12 2023-07-12 3-acyl-4-pyrone derivative as coupling reagent and application thereof in preparation of polypeptide and protein conjugate Pending CN117050046A (en)

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