CN111825742B - Polypeptide solid phase synthesis of CTPA as special coupling agent for amino acid ionic liquid - Google Patents

Polypeptide solid phase synthesis of CTPA as special coupling agent for amino acid ionic liquid Download PDF

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CN111825742B
CN111825742B CN201910312429.7A CN201910312429A CN111825742B CN 111825742 B CN111825742 B CN 111825742B CN 201910312429 A CN201910312429 A CN 201910312429A CN 111825742 B CN111825742 B CN 111825742B
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陈铭
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A polypeptide synthesis method using novel amino acid ionic liquid as reactant and N, N '-divinyl-N' -2-Chloroethyl Thiophosphamide (CTPA) as special coupling agent. The amino acid ionic liquid is an ionic liquid with amino acid with tert-butoxy protective groups as anions and imidazolium as cations. The polypeptide synthesis method mainly comprises the following steps: the preparation method comprises the steps of taking a preloaded resin for polypeptide synthesis as a starting material, CTPA as a coupling agent, and amino acid ionic liquid as a recoverable reactant, and performing amino acid coupling reaction to obtain the peptide compound. The synthesis method can complete the coupling reaction without alkali and other additive residues in 15 minutes at room temperature, consume less solvent, and rapidly synthesize the polypeptide product with high purity in high yield. The synthesis method disclosed by the invention is simple in process, environment-friendly and extremely high in practicability and sustainability.

Description

Polypeptide solid phase synthesis of CTPA as special coupling agent for amino acid ionic liquid
The technical field is as follows:
the invention relates to the technical field of biochemical synthesis, in particular to a polypeptide synthesis method using novel amino acid ionic liquid as a reactant and using N, N '-divinyl-N' -2-Chloroethyl Thiophosphamide (CTPA) as a special coupling agent.
The background technology is as follows:
in 2005, the Ohno group reported a series of entirely new room temperature ionic liquids, amino Acid Ionic Liquids (AAIL) [1,2]. Such ionic liquids may be prepared by neutralization of imidazolium hydroxide with 20 classes of natural amino acids. Although amino acid ionic liquids have long been used for liquid phase amide synthesis under heating [3], no controllable polypeptide synthesis method using amino acid ionic liquids has been used so far. Therefore, we are interested in studying: if the amino acid ionic liquid is used as a solvent and a reactant at the same time, the atom economy and the polypeptide synthesis efficiency can be improved. In view of the difficulty in extracting polypeptides from room temperature ionic liquid phases [4], we herein explore the utility of amino acid ionic liquids and their corresponding amino acid ionic liquid compatible coupling agents in Solid Phase Polypeptide Synthesis (SPPS). SPPS is an advantageous method of isolating the product and generally requires high concentrations of excess reactants and reagents to participate.
We note that multiple reactive groups of unprotected amino acid ionic liquids may lead to unwanted reactions in selective or multi-step organic syntheses. Thus, as with the use of protected amino acids in polypeptide chemistry, we expect that amino acid ionic liquids can also be used as reactants in polypeptide synthesis if the branching and N-terminus of the amino acid ionic liquid is also chemically protected. It is desirable that the protected amino acid ionic liquid, as well as the unprotected amino acid ionic liquid [1], may also be soluble in conventional organic solvents, thereby maximizing the reaction rate through high reactant concentrations. Thus, our research began with the development of a novel room temperature ionic liquid (Boc-AAIL) consisting of a 1-ethyl-3-methylimidazole cation ([ emim ]) and an amino acid anion ([ Boc-AA ]) protected by a tertiary butoxy and a different kind of branched protecting group.
The invention comprises the following steps:
in the present invention, we propose a unique coupling agent N, N '-divinyl-N' -2-Chloroethyl Thiophosphamide (CTPA), which is an impurity of the cancer drug thiopa, which has been found to be particularly suitable for solid phase peptide synthesis using Boc protected amino acid ionic liquids (Boc-AAILs). The CTPA mediated coupling reaction does not require activation and can be completed in a short 15 minutes without the addition of base and additives, resulting in a very good yield and excellent purity of the product.
Compared with the prior art, the polypeptide solid-phase synthesis method using CTPA as a special coupling agent for amino acid ionic liquid has the following innovation and advantages:
Boc-AAIL as a hundred-fold molar excess and ultra-high concentration reactant can be used to complete a solid phase reaction quickly without dissolution problems.
2. The use of CTPA significantly facilitates epimerized synthesis in AAIL without the addition of base and additives.
3. No pre-activation or prolonged reaction time is required.
4. All organosulfur compounds involved in the synthesis are odorless.
5. In addition, low solvent consumption and reuse of AAIL improves atomic economic efficiency.
Description of the drawings:
shown in fig. 1 is the amino acid ionic liquid Boc-AAIL prepared. The upper row is sequentially from left to right: [ emim ] [ Boc-Gly ], [ emim ] [ Boc-Ala ], [ emim ] [ Boc-Val ], [ emim ] [ Boc-Leu ], [ emim ] [ Boc-lle ], [ emim ] [ Boc-Phe ], [ emim ] [ Boc-Trp (For) ], [ emim ] [ Boc-Tyr (Bn) ], [ emim ] [ Boc-Asp (Bn) ] and [ emim ] [ Boc-His (Ts) ]. The next row is from left to right in turn: [ emim ] [ Boc-Asn ], [ emim ] [ Boc-Asn (Trt) ], [ emim ] [ Boc-Glu (Bn) ], [ emim ] [ Boc-Lys (Z) ], [ emim ] [ Boc-Gln ], [ emim ] [ Boc-Met ], [ emim ] [ Boc-Arg (Ts) ], [ emim ] [ Boc-Ser (Bn) ], [ emim ] [ Boc-Thr (Bn) ], [ emim ] [ Boc-Cys (Meb) ] and [ emim ] [ Boc-Pro ]. Wherein gly=glycine; ser=serine; ala = alanine; thr=threonine; val=valine; lle = isoleucyl; leu = leucine; tyr=tyrosine; phe = phenylalanine; his = histidine; pro = proline; asp=aspartic acid; met=methionine; glu = glutamic acid; trp=tryptophan; lys = lysine; cys = cysteine; arg=arginine. For = formyl; bn=benzyl; ts=tosyl; trt = trityl; z = phenoxycarbonyl; meb=p-methylbenzyl.
FIG. 2 is [ emim ]][Boc-Gly]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 3 is [ emim ]][Boc-Gly]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 4 is [ emim ]][Boc-Ala]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 5 is [ emim ]][Boc-Ala]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 6 is [ emim ]][Boc-Val]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 7 is [ emim ]][Boc-Val]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 8 is [ emim ]][Boc-Leu]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 9 is [ emim ]][Boc-Leu]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 10 is [ emim ]][Boc-lle]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 11 is [ emim ]][Boc-lle]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 12 is [ emim ]][Boc-Phe]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 13 is [ emim ]][Boc-Phe]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 14 is [ emim ]][Boc-Trp(For)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 15 is [ emim ]][Boc-Trp(For)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 16 is [ emim ]][Boc-Tyr(Bn)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 17 is [ emim ]][Boc-Tyr(Bn)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 18 is [ emim ]][Boc-Asp(Bn)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 19 is [ emim ]][Boc-Asp(Bn)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 20 is [ emim ]][Boc-His(Ts)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 21 is [ emim ]][Boc-His(Ts)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 22 is [ emim ]][Boc-Asn]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 23 is [ emim ]][Boc-Asn]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 24 is [ emim ]][Boc-Asn(Trt)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 25 is [ emim ]][Boc-Asn(Trt)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 26 is [ emim ]][Boc-Glu(Bn)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 27 is [ emim ]][Boc-Glu(Bn)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 28 is [ emim ]][Boc-Lys(Z)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 29 is [ emim ]][Boc-Lys(Z)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 30 is [ emim ]][Boc-Gln]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 31 is [ emim ]][Boc-Gln]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 32 is [ emim ]][Boc-Met]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 33 is [ emim ]][Boc-Met]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 34 is [ emim ]][Boc-Arg(Ts)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 35 is [ emim ]][Boc-Arg(Ts)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 36 is [ emim ]][Boc-Ser(Bn)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 37 is [ emim ]][Boc-Ser(Bn)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 38 is [ emim ]][Boc-Thr(Bn)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 39 is [ emim ]][Boc-Thr(Bn)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 40 is [ emim ]][Boc-Cys(Meb)]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 41 is [ emim ]][Boc-Cys(Meb)]13C nuclear magnetic resonance spectrum (100 MHz, DMSO-d) 6 )。
FIG. 42 is [ emim ]][Boc-Pro]1H nuclear magnetic resonance spectrum (400 MHz, DMSO-d) 6 )。
FIG. 43 is a 13C nuclear magnetic resonance spectrum of [ emim ] [ Boc-Pro ] (100 MHz, DMSO-d 6).
HPLC and mass spectrometry analysis of [ emim ] [ Boc-Asn ] after 15 minutes of CTPA treatment shown in FIG. 44 demonstrate the possibility of succinimide formation.
FIG. 45 is a schematic representation of AAIL [ emim ] [ Boc-Gly ], [ emim ] [ Boc-Ala ], [ emim ] [ Boc-Val ], [ emim ] [ Boc-Leu ], [ emim ] [ Boc-lle ], [ emim ] [ Boc-Phe ], [ emim ] [ Boc-Trp (For) ], [ emim ] [ Boc-Tyr (Bn) ], [ emim ] [ Boc-Asp (Bn) ], [ emim ] [ Boc-His (Ts) ], [ emim ] [ Boc-Asn ], [ emim ] [ Boc-Asn (Trt) ], FIGS.: RP-HPLC chromatograms of dipeptide products synthesized by [ emim ] [ Boc-Glu (Bn) ], [ emim ] [ Boc-Lys (Z) ], [ emim ] [ Boc-GIn ], [ emim ] [ Boc-Met ], [ emim ] [ Boc-Arg (Ts) ], [ emim ] [ Boc-Ser (Bn) ], [ emim ] [ Boc-Thr (Bn) ], [ emim ] [ Boc-Cys (Meb) ] and [ emim ] [ Boc-Pro ].
FIG. 46 shows a possible reaction mechanism of CTPA-mediated amide synthesis in Boc-AAIL.
The specific embodiment is as follows:
the experimental methods used in the following examples are conventional methods unless otherwise specified.
The experimental materials, reagents, etc. used in the examples described below are all commercially available or known experimental methods.
Example 1. Preparation and determination of novel amino acid ionic liquids Boc-AAIL.
Since the halide-anion exchange process is considered unsuitable for AAIL production, we pass through the process of [ emim ]][OH]Synthesis of Boc-AAIL [1] by simple neutralization with commercially available Boc-protected amino acids]. Pure Boc-AAIL is a clear, nearly colorless liquid at room temperature. These Boc-AAIL 1 The H NMR and elemental analysis results are consistent with the structures they present (see the figures). All Boc-AAIL are miscible with acetonitrile, methanol, dimethylformamide (DMF), partially miscible in water, but not miscible with diethyl ether at room temperature.
The thermophysical properties of Boc-AAIL were studied using thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC) (Table 1). It is evident from the measured values that, in addition to [ emim ]][Boc-Asn]Most Boc-AAIL decomposition starts at 73-105℃outside of 42℃decomposition (entry 11), entry 1-10, 12-21. DSC data shows that all Boc-AAIL have no melting point, but the glass transition temperature ranges from 42℃to 6 ℃. We found that using [ emim ]][Boc-Gly]And Boc-AAIL (entries 1-5, 16, 21) T involving small aliphatic or rigid side groups g The value is lower. Other Boc-AAIL containing aromatic rings (side chains or protecting groups) have a relatively high T g This may be due to the fragrance summation (item6，8-10，13，14，17-20)。[emim][Boc-Asn]，[emim][Boc-GIn]And [ emim ]][Boc-Trp(For)]T of (2) g Values near or above 0 ℃ indicate the presence of strong internal hydrogen bonding (entries 7, 11, 15). In addition to thermal properties, we measured the viscosity of Boc-AAIL with a capillary viscometer at 25 ℃. As shown in Table 1, a high eta value indicates that pure Boc-AAIL is unsuitable for use as a single solvent in solid phase synthesis, while fluidity (. Eta. '' -1 ) A significant improvement can be achieved by diluting Boc-AAIL with Dimethylformamide (DMF).
TABLE 1 thermal Properties and viscosity of Boc-AAIL and AAIL
a T g Glass transition temperature. b T d =thermal decomposition temperature, 5% mass loss was observed. c η = viscosity of pure Boc-AAIL at 25 ℃; viscosity of 80 mass% Boc-AAIL in DMF at η' =25 ℃.
EXAMPLE 2 Boc protective amino acid or Boc-AAIL solid phase coupling using common coupling agents.
In order to examine the actual efficiency of Boc-AAILs in SPPS, we first performed [ emim ]][Boc-Ala](2.0 ml, 80 mass% DMF solution) and H-L-phenalane-HMPB-Sample coupling reaction of the resin (100 mg,0.58mmol/g loading) was used as solid support. />The resin has excellent or swelling properties in DMF and ionic liquid (Table 2, entries 9-11) [5 ]]. After cleavage and purification from the resin with addition of hydrogen fluoride, the coupling reaction gives the product dipeptide L-Ala-L-Phe. For the purpose ofIn contrast, the coupling reaction was similarly carried out under standard conditions for Boc-SPPS using DMF (reagents 1-3) or [ bmim ]][PF6](1-butyl-3-methylimidazole hexafluorophosphate) as a solvent (items 5-7). EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and additive HOBt (hydroxybenzotriazole), HATU (1- [ bis (dimethylamino) methylene]-1H-1,2, 3-triazolo [4,5-b]Pyridinium 3-hexafluorophosphate oxide) and PyBOP (benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate) were chosen as optional coupling agents and DIEA (N, N-diisopropylethylamine) was used as base for all sample reactions. In [ bmim ]][PF 6 ]In the case of coupling in (clauses 5-7), both HATU and PyBOP were less efficient than the reactions achieved using the standard Boc strategy of DMF (clauses 1-3), and the application of EDC even resulted in complete failure of coupling. In contrast, HATU or PyBOP promoted reaction with AAIL occurred rapidly at the beginning (entries 10-11), but the reaction still did not give satisfactory yields after 1 hour. Screening studies showed that the efficiency of the coupling reagent decreased in the order HATU > PyBOP > EDC in the presence of imidazole-based ionic liquids. As a result of disappointing results, we therefore sought to find entirely new coupling reagents suitable for peptide synthesis with Boc-AAIL.
Example 3. Solid phase coupling of Boc protected amino acids or Boc-AAIL using CTPA.
In our previous drug stability studies, we observed CTPA, a degradation product of the chemotherapeutic drug thiopa, in amino acid ionic liquids [ emim][Ala]Unexpectedly forming a phenylalanine in the aqueous solution. Inspired by this finding, CTPA was used as a potential coupling agent in templating reactions. The results show, [ bmim][PF 6 ]CTPA-mediated peptide synthesis in (c) reached higher yields after 1 hour (table 2, entry 8), but severe epimerization occurred (17% d-Ala-L-Phe). As expected, no amide formation could be detected in the same synthesis reaction in DMF (table 2, entry 4). It is pleasant that [ emim ]][Boc-Ala]The coupling reaction was completed successfully within 15 minutes, the product HPLC purity was 99% and the yield was 91% without the need for base and additives. Upon the excitation of this successful example, we then extended the CTPA reactivity test to the applicationOther types of Boc-AAIL reactions. Wherein, except [ emim ]][Boc-Asn](entry 22), almost all Boc-AAIL performed well without detectable epimerization (entries 13-22, 24-32). We have found that the use of CTPA results in [ emim ]][Boc-Asn]Is converted into a succinimide derivative. However, this problem can be overcome by introducing a side chain protecting group trityl (entry 23). Notably, [ emim ]][Boc-GIn]Although having a AND [ emim ]][Boc-Asn]Similar carboxamide side chains, but without deamidation when treated with CTPA.
Table 2. Peptide synthesis by coupling Boc-amino acid or Boc-AAIL with Phe-ChemMatrix resin under various conditions.
a 4.0 equivalents of Boc-Ala-OH,8.0 equivalents of EDC,4.0 equivalents of HOBt,8.0 equivalents of DIEA,2.0 ml of DMF. b Isolation yield within 15 minutes. c Isolated yield within 1 hour. d 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of HATU,4.0 equivalents of HOBt,8.0 equivalents of DIEA. e 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of PyBOP,8.0 equivalents of DIEA,2.0 ml of DMF. f 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of CTPA,8.0 equivalents of DIEA,2.0 ml of DMF. g 4.0 equivalents of Boc-Ala-OH,8.0 equivalents of EDC,4.0 equivalents of HOBt,8.0 equivalents of DIEA,2.0 ml [ bmim ]][PF 6 ]。 h 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of HATU,4.0 equivalents of HOBt,8.0 equivalents of DIEA,2.0 ml [ bmim ]][PF 6 ]。 i 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of PyBOP,8.0 equivalents of DIEA,2.0 ml [ bmim ]][PF 6 ]。 j 4.0 equivalents of Boc-Ala-OH,4.0 equivalents of CTPA,8.0 equivalents of DlEA,2.0 ml [ bmim ]][PF 6 ]。 k 2.0 ml [ emim ]][Boc-Ala](80 mass percent DMF solution), 8.0 equivalents EDC,4.0 equivalents HOBt,8.0 equivalents DIEA. I 2.0 ml[emim][Boc-Ala](80 mass percent DMF solution), 4.0 equivalents HATU,4.0 equivalents HOBt,8.0 equivalents DIEA. m 2.0 ml [ emim ]][Boc-Ala](80 mass percent DMF solution, 2.0 ml), 4.0 eq PyBOP,8.0 eq DIEA. n 2.0 ml [ emim ]][Boc-Ala](80 mass percent DMF solution), 4.0 equivalents CTPA.
Example 4. Recovery and reuse of amino acid ionic liquid.
In fact, an important issue is that the high cost of excessive Boc-AAIL may hamper the commercial viability of CTPA-mediated polypeptide synthesis methods. Therefore, recycling of Boc-AAIL is necessary. After coupling, the liquid phase (FIG. 46) containing Boc-AAIL, DMF and the by-products N, N '-diethyl-N' -2-chloroethyl phosphoramide (scheme 1, 6) and imidazole-2-thione was separated from the solid phase and concentrated by evaporation. The resulting Boc-AAIL mixture was then extracted with diethyl ether, followed by saturated NaHCO 3 Extracting with aqueous solution. Recovery experiments showed that [ emim][Boc-Ala]At least 4 cycles can be performed in the template reaction without significant loss of activity.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc. are included in the scope of the present invention and the scope of disclosure.
Reference is made to:
[1]Fukumoto，K.；Yoshizawa，M.；Ohno，H.Room Temperature Ionic Liquids from 20Natural Amino Acids.J.Am.Chem.Soc.2005，127(8)，2398-2399.
[2](a)Ohno，H.；Fukumoto，K.Amino Acid Ionic Liquids.Acc.Chem.Res.2007，40(11)，1122-1129.(b)Kirchhecker，S.；Esposito，D.Amino Acid Based lonic Liquids：A Green and Sustainable Perspective.Curr.Opin.Green Sustain.Chem.2016，2，28-33.(c)Verma，C；Ebenso，E.；Quraishi，M.Recent Advancements in Synthesis and Applications of Amino Acid Ionic Liquids(AAILs)：A Mini Review.Clin.Med.Biochem.2018，4(3)，1-5.
[3]Furukawa，S.；Fukuyama，T.；Matsui，A.；Kuratsu，M.；Nakaya，R.；Ineyama，T.；Ueda，H.；Ryu，I.Coupling-Reagent-Free Synthesis of Dipeptides and Tripeptides Using Amino Acid Ionic Liquids.Chem.-A Eur.J.2015，21(34)，11980-11983.
[4]Vallette，H.；Ferron，L.；Coquerel，G.；Gaumont，A.-C.；Plaquevent，J.-C.Peptide Synthesis in Room Temperature Ionic Liquids.Tetrahedron Lett.2004，45(8)，1617-1619.
[5]Lawrenson，S.；North，M.；Peigneguy，F.；Routledge，A.Greener Solvents for Solid-Phase Synthesis.Green Chem.2017，19(4)，952-962.

Claims (1)

1. a dipeptide synthesis method is characterized in that amino acid ionic liquid with a tert-butoxy protective group is used as a reactant, and N, N '-divinyl-N' -2-chloroethyl thiophosphamide is used as a special coupling agent, wherein the structural formula of the N, N '-divinyl-N' -2-chloroethyl thiophosphamide is as follows:the amino acid ionic liquid with the tert-butoxy protective group is [ emim ]][Boc-Gly]，[emim][Boc-Ala]，[emim][Boc-Val]，
[emim][Boc-Leu]，[emim][Boc-Ile]，[emim][Boc-Phe]，[emim][Boc-Trp(For)]，
[emim][Boc-Tyr(Bn)]，[emim][Boc-Asp(Bn)]，[emim][Boc-His(Ts)]，[emim][Boc-Asn(Trt)]，[emim][Boc-Glu(Bn)]，[emim][Boc-Lys(Z)]，[emim][Boc-Gln]，[emim][Boc-Met]，
[emim][Boc-Arg(Ts)]，[emim][Boc-Ser(Bn)]，[emim][Boc-Thr(Bn)]，[emim][Boc-Cys(Meb)]And [ emim ]][Boc-Pro]One of said [ emim ]]Is 1-ethyl-3-methylimidazole cation, boc is tert-butoxycarbonyl, and For is formyl; bn is benzyl; the Ts is tosyl;the Trt is trityl; z is phenoxycarbonyl; meb is p-methylbenzyl, and the dipeptide synthesis method comprises the following basic steps: (1) Preparing amino acid ionic liquid with a tert-butoxy protective group by carrying out a neutralization reaction on 1-ethyl-3-methylimidazole hydroxide and Boc-protected amino acid; (2) 80 mass percent DMF solution of amino acid ionic liquid with tert-butoxy protective group and H-L-phenylalanine-HMPB-Mixing the resins according to the dosage proportion of 2mL of ionic liquid corresponding to each 100mg of the resins, and adding 4.0 times of molar equivalent of N, N '-divinyl-N' -2-chloroethyl thiophosphamide as a coupling agent to carry out coupling reaction; (3) After 15 minutes, the reaction is finished, and the reacted amino acid ionic liquid mixture and resin are subjected to solid phase separation; (4) Hydrogen fluoride is added to cut dipeptide from resin and purified to obtain corresponding product dipeptide; (5) Evaporating and concentrating the reacted amino acid ionic liquid mixture, and then using diethyl ether and saturated NaHCO to obtain the amino acid ionic liquid mixture 3 The aqueous solutions are respectively extracted to obtain the recovered amino acid ionic liquid with the tert-butoxy protective group, and the amino acid ionic liquid is reused for subsequent dipeptide synthesis.
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