CN103923016B - A kind of synthetic method of glycopeptide - Google Patents

A kind of synthetic method of glycopeptide Download PDF

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CN103923016B
CN103923016B CN201410092568.0A CN201410092568A CN103923016B CN 103923016 B CN103923016 B CN 103923016B CN 201410092568 A CN201410092568 A CN 201410092568A CN 103923016 B CN103923016 B CN 103923016B
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glycopeptide
ionic liquid
amino acid
fmoc
synthetic method
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CN103923016A (en
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李学兵
李长庚
张振兴
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Institute of Microbiology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C07D233/60—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms with hydrocarbon radicals, substituted by oxygen or sulfur atoms, attached to ring nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure

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Abstract

The invention discloses a kind of synthetic method of glycopeptide.The method comprises the steps: (1) Fmoc protected amino acid and ionic liquid obtain ion liquid supported unit by esterification; (2) described ion liquid supported unit is connected with any one monomer following, then sloughs described ionic liquid, obtain described glycopeptide; 1) glycosylated amino acid; 2) Fmoc protected amino acid described in glycosylated amino acid and at least one; Fmoc represents 9-fluorenylmethyloxycarbonyl.Tool of the present invention has the following advantages: ionic liquid is the small soluble molecules carrier that structure is clear and definite, and routine analysis means such as thin layer look spectrum ﹑ core magnetic therefore can be utilized to shake altogether the Real-Time Monitoring reaction process such as ﹑ mass spectrum; It is high that ionic liquid carrier also has stowage space, and be easy to synthesis, the advantages such as low price, therefore can realize a large amount of synthesis of glycopeptide.

Description

A kind of synthetic method of glycopeptide
Technical field
The present invention relates to a kind of synthetic method of glycopeptide, belong to organic chemistry filed.
Background technology
Glycopeptide is oligosaccharides and amino acid or the covalently bound and structural region formed of polypeptide chain in glycoprotein and proteoglycan, is biological information molecule very important in organism.In numerous biological phenomenas such as Xi born of the same parents Fen Lie ﹑ Xin Zhuan Dao ﹑ Xi born of the same parents Shi other ﹑ Ai Zheng ﹑ Mian Yi ﹑ Yan Zheng ﹑ infected by microbes and disease forming process, the very important role of the equal performer of glycopeptide.Relation between the structure and fuction of research glycopeptide plays an important role for the exploitation disclosing its biological function and sugared peptide medicament.Because the glycopeptide content in organism is low and have microheterogeneity, from biological sample, separation and purification glycopeptide is very difficult, usually need synthesis glycopeptide as Model Molecule to study its structure activity relationship, therefore the research of glycopeptide synthetic method becomes a large focus in glycobiology and carbohydrate chemistry field.
The chemical synthesis process of current glycopeptide mainly contains liquid phase synthesis and solid phase synthesis.Traditional liquid phase synthesis has the advantage of homogeneous reaction, but the separation and purification of product needs loaded down with trivial details column chromatography operation, wastes time and energy.The solid phase synthesis of glycopeptide mainly contains two kinds of strategies, and one is the glycopeptide synthesis strategy using previously prepared good glycosylated amino acid, can realize Fully automated synthesis; Two is carry out glycosylated glycopeptide synthesis strategy on the polypeptide or glycopeptide of solid phase carrier support.The great advantage of glycopeptide solid phase synthesis is that intermediate product washs and Purification by filtration by means of only simple, does not need loaded down with trivial details column chromatography operation.But the reaction in glycopeptide solid phase synthesis belongs to out-phase reaction, brings some defects thus.Under out-phase reaction system, low than in homogeneous system of the activity of reactant, usually needs to use greatly excessive reaction reagent to impel and reacts completely; Conventional analysis means can not be used as Real-Time Monitoring reaction process such as Bao layer Se Pu ﹑ He Ci Gong Zhen ﹑ mass spectrums; The stowage space of solid phase carrier is little, limits a large amount of synthesis of glycopeptide.Therefore, developing new glycopeptide synthetic technology, make it the advantage of the homogeneous reaction not only with liquid phase synthesis, the advantage that the out-phase also having solid phase synthesis concurrently is separated, is one of association area difficult point urgently to be resolved hurrily.
Ionic liquid (Ionicliquid, IL) is that one is in a liquid state in room temperature or close under room temperature, and the salt be made up of organic cation and organic or inorganic negatively charged ion, also claims low temperature molten salt.The designability of ionic liquid is strong, because the negatively charged ion and cationic selectable range forming it is large, therefore can as required, and the formation of simple change ion just can regulate and control its physics-chem characteristic such as fusing point, viscosity, density, solvability.Generally, ionic liquid has good thermostability, non-volatile, non-inflammability, does not explode and hypotoxicity, is thus eco-friendly in organic synthesis.
Summary of the invention
The object of this invention is to provide a kind of synthetic method of glycopeptide.
The present invention provide firstly a kind of ionic liquid, its structural formula such as formula shown in I,
Ionic liquid provided by the invention specifically prepares by following method: under nitrogen protection, using acetonitrile as solvent, and compound shown in formula 1, N-Methylimidazole and Potassium Hexafluorophosphate (KPF 6) be obtained by reacting, described reaction is carried out under the state of backflow;
The synthetic method of a kind of glycopeptide provided by the invention, comprises the steps:
(1) Fmoc protected amino acid and ionic liquid obtain ion liquid supported unit by esterification;
(2) described ion liquid supported unit is connected with any one monomer following, then sloughs described ionic liquid, obtain described glycopeptide;
1) glycosylated amino acid;
2) Fmoc protected amino acid described in glycosylated amino acid and at least one;
Fmoc represents 9-fluorenylmethyloxycarbonyl.
In above-mentioned synthetic method, described ionic liquid is ionic liquid shown in formula I.
In above-mentioned synthetic method, in step (1), described esterification is carried out under the effect of EDCI and DMAP;
Wherein, EDCI represents 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate;
DMAP represents DMAP.
In above-mentioned synthetic method, in step (2), described ion liquid supported unit and described monomer realize being connected by Fmoc deprotection reaction and polypeptide condensation successively;
Described ionic liquid is sloughed in the mixed solution of trifluoroacetic acid and water.
In above-mentioned synthetic method, described Fmoc deprotection reaction carries out under the effect of piperidines.
In above-mentioned synthetic method, described polypeptide condensation is carried out under the effect of HBTU, HOBt and DIPEA;
Wherein, HBTU represents benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate;
HOBt represents I-hydroxybenzotriazole;
DIPEA represents DIPEA.
In above-mentioned synthetic method, the structural formula of described glycopeptide such as formula shown in II,
In formula II, Ac represents ethanoyl; Fmoc represents 9-fluorenylmethyloxycarbonyl; Troc represents 2,2,2-trichloro-ethoxycarbonyl; Bzl represents benzyl; Ala represents L-Ala; Ser represents Serine; Val represents α-amino-isovaleric acid.
In above-mentioned synthetic method, the structural formula of described glycosylated amino acid such as formula shown in III,
In formula III, Ac represents ethanoyl; Fmoc represents 9-fluorenylmethyloxycarbonyl; Troc represents 2,2,2-trichloro-ethoxycarbonyl; TBS represents t-Butyldimethylsilyl.
Tool of the present invention has the following advantages:
(1) ionic liquid is the carrier of solubility, and therefore can realize homogeneous reaction, reaction efficiency is high, does not need to use excessive reaction reagent;
(2) solubleness of ionic liquid is controlled, therefore can realize out-phase and be separated, and ion liquid supported intermediate product is by means of only simple washing and filter purifying, does not need loaded down with trivial details column chromatography operation;
(3) ionic liquid is the small soluble molecules carrier that structure is clear and definite, and routine analysis means therefore can be utilized as Real-Time Monitoring reaction process such as Bao layer Se Pu ﹑ He Ci Gong Zhen ﹑ mass spectrums;
(4) also to have stowage space high for ionic liquid carrier, and be easy to synthesis, the advantages such as low price, therefore can realize a large amount of synthesis of glycopeptide.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The synthesis of embodiment 1, ionic liquid
Reaction equation is as follows:
The synthesis of compound 1: under nitrogen protection; 4-methylolphenol (4.00 grams, 32.22 mmole) ﹑ salt of wormwood (4.67 grams, 33.82 mmoles) and 1; 3-dibromopropane (9.85 milliliters, 96.44 mmoles) stirring and refluxing 5 hours in acetone (30 milliliters) solvent of drying.After thin-layer chromatography display 4-methylolphenol runs out of, by reacting liquid filtering and evaporated under reduced pressure solvent, gained mixture, through column chromatographic isolation and purification (ethyl acetate: sherwood oil=1:3 → 1:2), obtains compound 1(3.28 gram, 39.6%), be white solid.
The characterization result of compound 1 is as follows: 1hNMR (500MHz, CDCl 3) δ 7.29 (d, J=8.5Hz, 2H), 6.90 (d, J=8.5Hz, 2H), 4.62 (s, 2H), 4.11 (t, 2H), 3.61 (t, 2H), 2.32 (m, 2H); 13cNMR (150MHz, CDCl 3) δ 158.4,133.5,128.7,114.7,65.5,65.0,32.4,30.1.
The synthesis of ionic liquid shown in formula I: under nitrogen protection, compound 1(2.33 gram, 9.51 mmoles) be dissolved in dry acetonitrile (25 milliliters) solvent; add N-Methylimidazole (1.00 milliliters; 12.36 mmoles) and Potassium Hexafluorophosphate (KPF6,1.75 grams, 9.51 mmoles).Reaction system refluxes in 80 DEG C of stirred overnight, after thin-layer chromatography display compound 3 runs out of, reaction solution is cooled to room temperature, filter, evaporated under reduced pressure solvent, gained mixture washed with diethylether 3 to 4 times, obtains ionic liquid (3.54 grams shown in pure formula I, 95.0%), under normal temperature be colourless oil liquid.
Shown in formula I, the characterization result of ionic liquid is as follows: 1hNMR (400MHz, CD 3oD) δ 8.88 (s, 1H), 7.62 (s, 1H); 7.54 (s, 1H), 7.27 (d, J=8.0Hz; 2H), 6.87 (d, J=8.0Hz, 2H); 4.52 (s, 2H), 4.43 (t, J=6.8Hz; 2H), 4.05 (t, J=5.6Hz, 2H); 3.90 (s, 3H), 2.36 (m, 2H); 13cNMR (150MHz, CD 3oD) δ 159.2,137.7,135.2,129.7,124.8,123.7,115.3,65.5,64.8,48.2,36.4,30.6; HR-ESIMS:m/z247.14435 [M-PF 6] +(calcd247.14410 [M-PF 6] +forC 14h 19n 2o 2).
The synthesis of embodiment 2, glycosylated amino acid construction unit formula III
Reaction equation is as follows:
In above-mentioned reaction equation, the implication of the abbreviation used is as follows: Troc:2,2,2-trichloro-ethoxycarbonyl; TBSCl: TERT-BUTYL DIMETHYL CHLORO SILANE; TBS: t-Butyldimethylsilyl; Ac: ethanoyl; Fmoc:9-fluorenylmethyloxycarbonyl; Ser: Serine; Allyl: allyl group; DMF:N, dinethylformamide; DCM: methylene dichloride; DBU:1,8-diazabicylo 11 carbon-7-alkene.
" one kettle way " synthesizes glycosyl donor 3: compound 2(12.0 gram, 33.84 mmoles) be dissolved in pyridine (150 milliliters) solvent, stir and be cooled to 0 DEG C, by TERT-BUTYL DIMETHYL CHLORO SILANE (6.63 grams, 44.00 mmoles) join in reaction system, after 30 minutes, in room temperature for overnight.After thin-layer chromatography display compound 5 runs out of, diacetyl oxide (35 milliliters) is added in reaction system, in stirred at ambient temperature 6 hours, evaporated under reduced pressure solvent, products therefrom is dissolved in methylene dichloride (300 milliliters), wash by the saturated NaHCO3 of 1MHCl ﹑ and saturated NaCl extraction successively, organic phase anhydrous Na 2sO 4dry.Cross and filter anhydrous Na 2sO 4after, by organic phase evaporated under reduced pressure solvent, after gained compound vacuumizes 1 hour, then be dissolved in DMF (50 milliliters), add acetic acid hydrazine (3.74 grams, 40.61 mmoles), in stirred at ambient temperature 2 hours.Then in reaction solution, add methylene dichloride (300 milliliters), wash by saturated NaCl extraction, organic phase anhydrous Na 2sO 4dry.Cross and filter anhydrous Na 2sO 4after, by organic phase evaporated under reduced pressure solvent, after gained compound vacuumizes 1 hour, be dissolved in again in dry methylene dichloride (100 milliliters), add Trichloroacetonitrile (16 milliliters) and 1,8-diazabicylo 11 carbon-7-alkene (DBU successively, 1.0 milliliters, 6.77 mmoles).In stirred at ambient temperature after 3 hours, evaporated under reduced pressure solvent, gained mixture is through column chromatographic isolation and purification (ethyl acetate: sherwood oil=1:7), and obtain compound 3(16.28 gram, four step productive rates are 69%), be white foam solid.
The characterization result of compound 3 is as follows: 1hNMR (400MHz, CDCl 3) δ 8.75 (s, 1H), 6.43 (d, J=3.6Hz, H-1; 1H), 5.34 (t, 1H), 5.25 (t, 1H); 5.16 (d, J=9.2Hz, 1H), 4.72 (s, 2H); (4.23 td, 1H), 3.99 (m, 1H), 3.71 (q; 2H), 2.05 (d, 6H), 0.87 (s, 9H); 0.03 (s, 3H), 0.03 (s, 3H); 13cNMR (125MHz, CDCl 3) δ 171.4,169.2,160.5,154.3,95.4,94.8,90.9,74.7,73.1,70.8,68.0,61.8,54.0,25.9,20.8,20.8,18.3 ,-5.3 ,-5.4.
The synthesis of compound 4: glycosyl donor 3(5.10 gram, 7.32 mmoles) and Fmoc-L-Ser-OAllyl(2.24 gram, 6.10 mmoles) vacuumize 2 hours after, under nitrogen protection, add activation molecular sieve (3.0 grams) and dry methylene dichloride (50 milliliters).Reaction system is cooled to-30 DEG C, the trifluoromethanesulfonic acid (53 microlitre) be dissolved in dry methylene dichloride (5 milliliters) is slowly added drop-wise in reaction system, after stirring 1 hour in-30 DEG C, after thin-layer chromatography display reacts completely, add triethylamine (80 microlitre) termination reaction, cross and filter molecular sieve, evaporated under reduced pressure solvent, gained mixture, through column chromatographic isolation and purification (ethyl acetate: sherwood oil=1:4), obtains compound 4(4.68 gram, 85%), be white foam solid.
The characterization result of compound 4 is as follows: 1hNMR (400MHz, CDCl 3) δ 7.78 (d, 2H), 7.64 (q, 2H); (7.41 t, 2H), 7.34 (q, 2H); (5.90 m, 1H), 5.81 (d, 1H); (5.34 d, 1H), 5.26 (d, 1H); (5.20 t, 1H), 5.14 (d, 1H); (5.02 t, 1H), 4.76 (d, 1H); 4.68 (d, 2H), 4.57-4.46 (m; 4H), 4.26-4.23 (m, 3H); 3.88 (d, 1H), 3.74-3.59 (m; 3H), 3.50 (m, 1H); 2.03 (s, 6H), 0.89 (s; 9H), 0.05 (s, 6H); 13cNMR (125MHz, CDCl 3) δ 171.0,169.5,169.4,156.3,154.4,144.1; 143.7,141.5,141.4,131.5,127.9,127.3; 125.5,125.3,120.2,118.9,100.7,95.5; 74.9,74.5,72.4,69.1,69.0,67.4; 66.5,62.5,56.3,54.1,47.3,25.9; 20.9,20.8,18.4 ,-5.2 ,-5.3; HR-ESIMS:m/z923.2240 [M+Na] +(calcd923.2234 [M+Na] +forC 40h 51cl 3n 2naO 13si).
The synthesis of glycosylated amino acid construction unit formula III: compound 4(4.68 gram, 5.19 mmoles) be dissolved in tetrahydrofuran (THF) (50 milliliters), add tetrakis triphenylphosphine palladium (0) (300 milligrams successively, 0.26 mmole) and methylphenylamine (2.25 milliliters, 20.75 mmoles), in stirred at ambient temperature 1 hour.After thin-layer chromatography display compound 4 runs out of, evaporated under reduced pressure solvent, gained mixture, through column chromatographic isolation and purification (methylene dichloride: methyl alcohol=30:1), obtains glycosylated amino acid shown in formula III (4.02 grams, 90%), is pale yellow foam solid.
The characterization result of formula III compound is as follows: 1hNMR (500MHz, acetone-d 6) δ 7.87 (d, 2H), 7.74 (t, 2H); (7.42 t, 2H), 7.35 (m, 2H); 6.98 (d, J=9.0Hz, 1H), 6.49 (d; J=8.5Hz, 1H), 5.27 (t, 1H); (5.06 t, 1H), 4.89 (q, 2H); (4.54 m, 2H), 4.41 (m, 1H); (4.27 m, 3H), 4.02 (dd, 1H); (3.83 dd, 1H), 3.74 (q, 1H); 3.67 (m, 2H), 1.98 (s; 3H), 1.93 (s, 3H); 0.90 (s, 9H), 0.09 (s; 3H), 0.07 (s, 3H); 13cNMR (125MHz, acetone-d 6) δ 171.3,170.5,169.7,157.0,155.5,145.0,144.9,142.1,128.6,128.0; 126.3,126.2,120.8,101.5,96.9,75.3,74.8,73.8,69.7,67.6; 62.9,56.9,54.8,47.9,26.2,20.7,18.9 ,-5.1 ,-5.2; HR-ESIMS:m/z883.1937 [M+Na] +(calcd883.1931 [M+Na] +forC 37h 47cl 3n 2naO 13si).
Glycopeptide shown in embodiment 3, synthesis type II
In above-mentioned flow process, each step reaction condition is as follows: (a) 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCI), DMAP (DMAP), acetonitrile, nitrogen, room temperature, 7 hours; (b) piperidines, methylene dichloride, room temperature, 0.5 hour; (c) benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU), I-hydroxybenzotriazole (HOBt), DIPEA (DIPEA), acetonitrile, nitrogen, room temperature, 8 hours; (d) trifluoroacetic acid: water=95:5 (v/v), methylene dichloride, 1 hour.
In following preparation, the out-phase separation method used is as follows:
For the compound that purifying is ion liquid supported.Concrete operations flow process is: reaction gained mixture is dissolved in methylene dichloride (10 mls/g), add the isopropyl ether of 4 times of volumes, then Rotary Evaporators removal of solvent under reduced pressure is utilized, treat that the cumulative volume of residual solvent is about a half of original bulk volume, to stop the rotation evaporation, methylene dichloride now in system is all removed, ion liquid supported compound all precipitates, by collected by filtration and with washed with diethylether precipitation, gained precipitation be pure ion liquid supported compound.
In following preparation, the Fmoc deprotection method used is as follows:
For removing amino protecting group Fmoc(9-fluorenylmethyloxycarbonyl).Concrete operations flow process is: the compound of Fmoc protection is dissolved in methylene dichloride (15 mls/g); add the piperidines of 10 equivalents; in stirred at ambient temperature 0.5 hour; after thin-layer chromatography display reacts completely; evaporated under reduced pressure liquid; gained mixture out-phase separation method is purified, and obtains the de-protected product of pure amino.
In following preparation, the polypeptide method of condensing used is as follows:
For amino acid or the amino of polypeptide and the condensation of carboxyl, form the amido linkage in peptide main chain.Concrete operations flow process is: under nitrogen protection, glycosylated amino acid (1.5 equivalent) ﹑ benzotriazole-N shown in the amino acid (2 equivalent) of amino de-protectedization He Wu ﹑ Fmoc protection or formula III, N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU, 2 or 1.6 equivalents) and I-hydroxybenzotriazole (HOBt, 2 or 1.6 equivalents) be dissolved in dry acetonitrile solvent, by N, N-diisopropylethylamine (DIPEA, 2.5 or 2 equivalents) drop in reaction system, reaction solution was in stirred at ambient temperature 8 hours, after thin-layer chromatography display reaction safety, evaporated under reduced pressure solvent, be dissolved in again in methylene dichloride, use the saturated NaHCO of 1MHCl ﹑ successively 3and saturated NaCl extraction is washed, organic phase anhydrous Na 2sO 4drying, filter, solvent evaporated, gained mixture out-phase separation method is purified, and obtains pure ion liquid supported product.
Esterification (loading) method: first amino acid construction unit is propped up by esterification and is downloaded on ionic liquid carrier, as follows:
The synthesis of chemical compounds I a: under nitrogen protection; ionic liquid carrier type I (1.50 grams; 3.82 mmole) ﹑ Fmoc-L-Ala-OH(2.26 gram; 7.27 mmole) ﹑ 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCI; 1.47 gram; 7.65 mmoles) and DMAP (DMAP, 93 milligrams, 0.76 mmole) be dissolved in dry acetonitrile (25 milliliters).Reaction solution was in stirred at ambient temperature 7 hours, and thin-layer chromatography display reacts completely, evaporated under reduced pressure solvent, then is dissolved in methylene dichloride (200 milliliters), uses the saturated NaHCO of 1MHCl ﹑ successively 3and saturated NaCl extraction is washed, organic phase anhydrous Na 2sO 4drying, filter, solvent evaporated, gained mixture out-phase separation method is purified, and obtains pure chemical compounds I a(2.50 gram, 95.4%), be white foam solid.
The characterization result of chemical compounds I a is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.25 (s, 1H), 7.86 (d, 2H); 7.78 (s, 1H), 7.69 (s, 3H); 7.41 (t, 2H), 7.31 (q; 4H), 6.98 (d, 1H); 6.84 (d, 2H), 5.08 (s; 2H), 4.57 (s, 2H); 4.34-4.25 (m, 3H), 4.20 (d; 1H), 4.07 (s, 2H); 4.02 (s, 3H), 2.41 (s; 2H), 1.41 (d, 3H); 13cNMR (125MHz, acetone-d 6) δ 173.5,159.4,156.8,145.1,144.9,142.1,137.9,130.7,129.5,128.5,127.9,126.2,126.1,124.7,123.6,120.8,115.2,67.2,66.8,65.3,50.7,47.9,36.6,30.3,17.8; ESI-MS:m/z540.2 [M-PF 6] +; HR-ESIMS:m/z540.2489 [M-PF 6] +(calcd540.2493 [M-PF 6] +forC 32h 34n 3o 5).
The synthesis of compoundsⅰb: adopt Fmoc deprotection method, uses piperidines (3.6 milliliters, 36.5 mmoles) to remove chemical compounds I a(2.50 gram, 3.65 mmoles) Fmoc protecting group.The amino de-protected compound of gained; adopt polypeptide method of condensing; with Fmoc-L-Ser (Bzl)-OH(3.04 gram; 7.29 mmole) ﹑ benzotriazole-N; N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU; 2.77 gram; 7.29 mmole) ﹑ I-hydroxybenzotriazole (HOBt, 0.99 gram, 7.29 mmole) ﹑ N; N-diisopropylethylamine (DIPEA; 1.56 milliliters, 9.12 mmoles) and the acetonitrile (50 milliliters) of drying, be used to synthetic compound I b(2.84 gram; 90.4%), be white foam solid.
The characterization result of compoundsⅰb is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.12 (s, 1H), 7.86 (d, 3H), 7.72 (t; 4H), 7.42-7.30 (m, 11H), 6.85 (d, 3H); (5.06 s, 2H), 4.55-4.50 (m, 6H), 4.32 (m; 2H), 4.23 (t, 1H), 4.07 (s, 2H); (4.00 s, 3H), 3.78 (d, 2H); (2.40 s, 2H), 1.37 (d, 3H); 13cNMR (125MHz, acetone-d 6) δ 173.0,170.4,159.3,157.0,145.0,142.0; 139.3,137.8,130.7,129.4,129.1,128.9; 128.5,128.4,128.3,128.0,126.2; 124.7,123.6,120.8,115.2,73.5; 71.1,67.4,66.9,65.2,55.9; 49.1,47.9,36.6,30.3,17.9; ESI-MS:m/z717.3 [M-PF 6] +; HR-ESIMS:m/z717.3282 [M-PF 6] +(calcd717.3283 [M-PF 6] +forC 42h 45n 4o 7).
The synthesis of chemical compounds I c: adopt Fmoc deprotection method, uses piperidines (0.91 milliliter, 9.3 mmoles) to remove the Fmoc protecting group of compoundsⅰb (0.80 gram, 0.93 mmole).The amino de-protected compound of gained; adopt polypeptide method of condensing; with glycosylated amino acid construction unit formula III (1.20 grams; 1.39 mmole) ﹑ benzotriazole-N; N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU; 0.56 gram; 1.48 mmole) ﹑ I-hydroxybenzotriazole (HOBt, 0.20 gram, 1.48 mmole) ﹑ N; N-diisopropylethylamine (DIPEA; 0.32 milliliter, 1.85 mmoles) and the acetonitrile (40 milliliters) of drying, be used to synthetic compound I c(1.07 gram; 78%), be pale yellow foam solid.
The characterization result of chemical compounds I c is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.13 (s, 1H), 7.87 (d, 2H), 7.83 (s, 1H), 7.74-7.66 (m, 4H), 7.63 (d, 1H), 7.42 (t, 2H), 7.35-7.25 (m, 9H), 7.11 (d, 1H), 6.87 (d, 2H), 6.78 (d, 1H), 5.27 (t, 1H), 5.08 (s, 2H), 5.01 (t, 1H), 4.89 (t, 2H), 4.61 (t, 3H), 4.57-4.48 (m, 5H), 4.37-4.24 (m, 4H), 4.12 (t, 2H), 4.06 (s, 4H), 3.86-3.80 (m, 2H), 3.76-3.69 (m, 4H), 2.45 (m, 2H), 1.98 (s, 3H), 1.92 (s, 3H), 1.37 (d, 3H), 0.88 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H), 13cNMR (125MHz, acetone-d 6) δ 173.0,170.5,170.3,170.2,169.8,159.3,157.3, 155.6,142.1,142.0,130.7,129.1,128.6,128.4, 128.3,128.0,124.8,123.6,120.8,115.2,101.6, 97.0,75.2,74.6,73.6,69.8,67.7,66.9, 65.3,62.9,56.9,55.5,49.2,48.0,47.9, 36.7,26.3,20.8,18.9,17.8 ,-5.1 ,-5.2, ESI-MS:m/z1339.4 [M-PF 6] +(calcd1339.4 [M-PF 6] +).
The synthesis of chemical compounds I d: adopt Fmoc deprotection method, uses piperidines (0.71 milliliter, 7.2 mmoles) to remove chemical compounds I c(1.07 gram, 0.72 mmole) Fmoc protecting group.The amino de-protected compound of gained; adopt polypeptide method of condensing; with Fmoc-L-Val-OH(0.49 gram; 1.44 mmole) ﹑ benzotriazole-N; N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU; 0.55 gram; 1.44 mmole) ﹑ I-hydroxybenzotriazoles (HOBt, 0.20 gram, 1.48 mmole) ﹑ N; N-diisopropylethylamine (DIPEA; 0.31 milliliter, 1.80 mmoles) and the acetonitrile (20 milliliters) of drying, be used to synthetic compound I d(1.01 gram; 89%), be pale yellow foam solid.
The characterization result of chemical compounds I d is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.17 (s, 1H), 7.87 (s, 2H), 7.83 (s, 1H), 7.74 (s, 4H), 7.58 (m, 2H), 7.42 (t, 2H), 7.36-7.30 (m, 8H), 7.28-7.24 (m, 2H), 6.96 (d, 1H), 6.88 (d, 2H), 5.25 (t, 1H), 5.08 (s, 2H), 4.97 (q, 2H), 4.74-4.61 (m, 6H), 4.56-4.34 (m, 6H), 4.27 (t, 1H), 4.13 (t, 2H), 4.08-4.01 (m, 4H), 3.91 (m, 1H), 3.84 (q, 1H), 3.76-3.67 (m, 4H), 3.50 (m, 1H), 2.45 (m, 2H), 2.16 (m, 1H), 1.89 (s, 6H), 1.41 (d, 3H), 0.99 (t, 6H), 0.86 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H), 13cNMR (125MHz, acetone-d 6) δ 172.9,172.4,170.4,170.1,169.8,159.3,157.7,155.5,145.0, 144.8,142.1,139.2,137.7,130.8,129.5,129.1,128.6,128.5, 128.4,128.3,128.0,127.9,126.3,124.8,123.6,120.8,115.2, 102.8,97.1,75.2,74.9,74.0,73.6,70.5,69.7,67.5, 66.9,65.3,62.9,62.2,56.9,54.3,54.1,49.2,48.0, 36.7,26.3,20.7,19.9,18.8,17.9 ,-5.1 ,-5.2, ESI-MS:m/z1438.5 [M-PF 6] +(calcd1438.5 [M-PF 6] +).
The synthesis of chemical compounds I e: adopt Fmoc deprotection method, uses piperidines (0.60 milliliter, 6.1 mmoles) to remove chemical compounds I d(0.96 gram, 0.61 mmole) Fmoc protecting group.The amino de-protected compound of gained; adopt polypeptide method of condensing; with Fmoc-L-Ala-OH(0.38 gram; 1.21 mmole) ﹑ benzotriazole-N; N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU; 0.46 gram; 1.21 mmole) ﹑ I-hydroxybenzotriazoles (HOBt, 0.16 gram, 1.21 mmole) ﹑ N; N-diisopropylethylamine (DIPEA; 0.26 milliliter, 1.52 mmoles) and the acetonitrile (20 milliliters) of drying, be used to synthetic compound I e(0.95 gram; 95%), be pale yellow foam solid.
The characterization result of chemical compounds I e is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.17 (s, 1H), 7.87 (d, 2H), 7.84 (s, 1H), 7.74-7.72 (m, 4H), 7.68 (d, 1H), 7.61 (d, 1H), 7.55 (d, 1H), 7.42 (t, 3H), 7.37-7.26 (m, 10H), 6.88 (d, 2H), 5.30 (t, 1H), 5.08 (s, 2H), 5.00-4.97 (m, 2H), 4.79 (d, 1H), 4.68-4.46 (m, 9H), 4.38-4.33 (m, 3H), 4.26 (t, 2H), 4.14 (t, 2H), 4.06 (s, 3H), 3.93 (m, 1H), 3.85 (q, 1H), 3.79-3.69 (m, 4H), 3.65 (m, 1H), 2.46 (m, 2H), 2.17 (m, 1H), 1.96 (s, 3H), 1.92 (s, 3H), 1.41 (d, 6H), 0.94 (m, 6H), 0.87 (s, 9H),-0.05 (s, 3H),-0.04 (s, 3H), 13cNMR (125MHz, acetone-d 6) δ 174.4,172.9,172.2,170.5,170.1,169.8, 159.3,157.2,155.5,145.1,144.9,142.1, 139.2,137.7,130.8,129.6,129.1,128.6, 128.4,128.3,128.0,126.2,124.8,123.6, 120.8,115.2,102.5,97.1,75.3,74.9, 73.9,73.6,70.5,70.1,69.9,67.5, 66.9,65.3,62.9,60.0,56.9,54.5, 54.1,51.8,49.2,48.0,47.9, 36.7,30.6,26.3,20.8,19.8, 18.8,18.6,17.8 ,-5.1 ,-5.2, MALDI-TOF-MS:m/z1509.55 [M-PF 6] +(calcd1509.54 [M-PF 6] +).
The synthesis of compound 5: adopt Fmoc deprotection method, uses piperidines (0.55 milliliter, 5.5 mmoles) to remove chemical compounds I e(0.90 gram, 0.55 mmole) Fmoc protecting group.The amino de-protected compound of gained; adopt polypeptide method of condensing; with Fmoc-L-Ser (Bzl)-OH(0.46 gram; 1.09 mmole) ﹑ benzotriazole-N; N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU; 0.42 gram; 1.09 mmole) ﹑ I-hydroxybenzotriazole (HOBt, 0.15 gram, 1.09 mmole) ﹑ N; N-diisopropylethylamine (DIPEA; 0.24 milliliter, 1.37 mmoles) and the acetonitrile (20 milliliters) of drying, be used to synthetic compound 5(0.92 gram; 92%), be pale yellow foam solid.
The characterization result of compound 5 is as follows: 1hNMR (500MHz, acetone-d 6) δ 9.16 (s, 1H), 8.07 (d, 1H), 7.87 (d, 2H), 7.83 (s, 1H), 7.74-7.70 (m, 4H), 7.58-7.53 (m, 3H), 7.49 (d, 1H), 7.43-7.25 (m, 16H), 7.02 (d, 1H), 6.87 (d, 2H), 5.29 (t, 1H), 5.08 (s, 2H), 5.02-4.96 (m, 2H), 4.83 (d, 1H), 4.65-4.44 (m, 12H), 4.32-4.23 (m, 3H), 4.14 (t, 2H), 4.05 (s, 3H), 3.93-3.65 (m, 11H), 2.46 (m, 2H), 2.17 (m, 1H), 1.94 (s, 3H), 1.90 (s, 3H), 1.40 (d, 3H), 1.37 (d, 3H), 0.92 (d, 6H), 0.87 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H), 13cNMR (125MHz, acetone-d 6) δ 174.1,172.9,172.4,170.6,170.1,169.8, 159.3,157.5,155.5,144.9,142.1,139.2, 139.1,137.7,130.7,130.0,129.1,128.6, 128.4,128.2,128.0,126.2,126.1,124.8, 123.6,120.8,115.2,102.1,97.1, 75.3,74.8,74.0,73.6,73.5, 70.6,69.9,67.6,66.9,65.3, 62.9,56.9,54.2,49.2,48.0, 47.9,36.7,30.6,26.3,20.7, 19.8,18.9,17.8 ,-5.1 ,-5.2, ESI-MS:m/z1686.6 [M-PF 6] +(calcd1686.6 [M-PF 6] +).
The synthesis of glycopeptide shown in formula II: compound 5(86 milligram, 0.0469 mmole) be dissolved in methylene dichloride (2 milliliters), then add trifluoroacetic acid: water=95:5(v/v) mix reagent (0.5 milliliter).Reaction solution was in stirred at ambient temperature 1 hour, and evaporated under reduced pressure liquid, gained mixture uses high performance liquid chromatography (HPLC) separation and purification.Separation condition is: AgilentEclipseXDB-C18 (5 μm, 9.4 × 250mm) chromatographic column, gradient elution 25 minutes, and eluent is the methyl alcohol (trifluoroacetic acid containing 0.1%) of 80% → 90%, and flow velocity is 3 ml/min.Obtaining glycopeptide (37 milligrams, 59%) shown in pure formula II, is white solid.
Shown in formula II, the characterization result of glycopeptide is as follows: 1hNMR (500MHz, DMSO-d 6) δ 12.56 (s, 1H), 8.21 (d, J=6.5Hz, 1H), 8.18 (d, J=7.5Hz, 1H), 7.96 (d, J=7.5Hz, 1H), 7.89 (d, J=7.5Hz, 2H), 7.78 (d, J=9.5Hz, 1H), 7.75-7.71 (m, 4H), 7.65 (d, J=8.5Hz, 1H), 7.41 (t, 2H), 7.34-7.24 (m, 12H), 5.58 (d, J=6.0Hz, 1H), 4.93 (d, J=12.0Hz, 1H), 4.89 (t, J=10Hz, 1H), 4.61-4.58 (m, 2H), 4.55-4.43 (m, 6H), 4.40-4.33 (m, 2H), 4.30-4.19 (m, 6H), 4.13 (dd, 1H), 3.73 (m, 2H), 3.64-3.51 (m, 5H), 3.41-3.35 (m, 2H), 2.01 (s, 3H), 1.97 (m, 1H), 1.93 (s, 3H), 1.29 (d, J=7.5Hz, 3H), 1.21 (d, J=7.0Hz, 3H), 0.79 (d, J=7.0Hz, 3H), 0.75 (d, J=6.5Hz, 3H), 13cNMR (125MHz, DMSO-d 6) δ 173.8,171.9,170.8,170.4,169.6,169.3,168.9,168.7,156.0, 154.3,143.9,143.8,140.7,138.2,128.2,127.7,127.5, 127.4,127.1,125.4,120.1,100.9,96.3,75.4,73.5, 73.3,72.1,72.0,69.9,69.8,69.3,67.9,65.8, 63.1,57.3,55.5,54.7,52.8,52.6,48.1,47.7, 46.6,30.8,20.8,20.6,19.2,18.0,17.7,17.4, HR-ESIMS:m/z1364.39155 [M+Na] +(calcd1364.39464 [M+Na] +forC 62h 74cl 3n 7naO 20).

Claims (8)

1. an ionic liquid, its structural formula such as formula shown in I,
2. a synthetic method for glycopeptide, comprises the steps:
(1) Fmoc protected amino acid and ionic liquid obtain ion liquid supported unit by esterification;
(2) described ion liquid supported unit is connected with any one monomer following, then sloughs described ionic liquid, obtain described glycopeptide;
1) glycosylated amino acid;
2) the glycopeptide monomer that forms of Fmoc protected amino acid described in glycosylated amino acid and at least one;
Fmoc represents 9-fluorenylmethyloxycarbonyl;
Described ionic liquid is ionic liquid shown in claim 1 Chinese style I.
3. synthetic method according to claim 2, is characterized in that: in step (1), described esterification is carried out under the effect of EDCI and DMAP;
Wherein, EDCI represents 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate;
DMAP represents DMAP.
4. synthetic method according to claim 2, is characterized in that: in step (2), and described ion liquid supported unit and described monomer realize being connected by Fmoc deprotection reaction and polypeptide condensation successively;
Described ionic liquid is sloughed in the mixed solution of trifluoroacetic acid and water.
5. synthetic method according to claim 4, is characterized in that: described Fmoc deprotection reaction carries out under the effect of piperidines.
6. synthetic method according to claim 4, is characterized in that: described polypeptide condensation is carried out under the effect of HBTU, HOBt and DIPEA;
Wherein, HBTU represents benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate;
HOBt represents I-hydroxybenzotriazole;
DIPEA represents DIPEA.
7. synthetic method according to claim 2, is characterized in that: the structural formula of described glycopeptide such as formula shown in II,
In formula II, Ac represents ethanoyl; Fmoc represents 9-fluorenylmethyloxycarbonyl; Troc represents 2,2,2-trichloro-ethoxycarbonyl; Bzl represents benzyl; Ala represents L-Ala; Ser represents Serine; Val represents α-amino-isovaleric acid.
8. synthetic method according to claim 2, is characterized in that: the structural formula of described glycosylated amino acid such as formula shown in III,
In formula III, Ac represents ethanoyl; Fmoc represents 9-fluorenylmethyloxycarbonyl; Troc represents 2,2,2-trichloro-ethoxycarbonyl; TBS represents t-Butyldimethylsilyl.
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