CN115340472B - Glutamic acid derivative and synthesis method and application thereof - Google Patents

Glutamic acid derivative and synthesis method and application thereof Download PDF

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CN115340472B
CN115340472B CN202211135753.4A CN202211135753A CN115340472B CN 115340472 B CN115340472 B CN 115340472B CN 202211135753 A CN202211135753 A CN 202211135753A CN 115340472 B CN115340472 B CN 115340472B
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glutamic acid
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acid derivative
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butyl ester
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CN115340472A (en
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李宜明
王容天
王玉
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Hefei University of Technology
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    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C269/06—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups by reactions not involving the formation of carbamate groups
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    • C07—ORGANIC CHEMISTRY
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • 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
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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Abstract

The invention discloses a glutamic acid derivative, a synthesis method and application thereof, wherein the molecular structure of the glutamic acid derivative is shown as follows: The invention firstly carries out substitution reaction on commercially purchased N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester and alpha-bromo-4-methoxyacetophenone, and then removes tert-butyl ester serving as carboxyl protecting group of main glutamic acid chain, thus obtaining the glutamic acid derivative with photosensitive groups on side chains. The glutamic acid derivative disclosed by the invention can be compatible with polypeptide solid-phase synthesis and is used for preparing and producing various polypeptides requiring light-operated protecting groups.

Description

Glutamic acid derivative and synthesis method and application thereof
Technical Field
The invention relates to a glutamic acid derivative compatible with solid phase synthesis and provided with a photosensitive p-methoxybenzoyl group on a side chain, and a synthesis method and application thereof, and belongs to the technical field of protein synthesis.
Background
The biological functions of the light cage peptide and the protein are inhibited by the photocleavable protecting group, and the activity of the light cage peptide and the protein can be recovered under the irradiation of light. These compounds are widely used to detect and elucidate complex biological processes because illumination can be precisely controlled in a non-invasive manner in terms of space, time and amplitude. Side chain cage forms of serine, threonine, tyrosine, cysteine, lysine, arginine, glutamine, aspartic acid, and glutamic acid (Glu) have been reported. Among these functions, carboxylic acid groups play an important role in protein activity and protein interactions with other biological macromolecules or small molecules. Peptide and protein derivatives that are carboxylic acid moiety-entrapped have been developed to study biochemical mechanisms and protein-protein/ligand interactions.
For example, the article (J.am.chem.Soc.1991, 113, 2758-2760.) reports a method for triggering an enzymatic reaction by photoactivatable phage T4 lysozyme containing aspartyl b-nitrobenzyl ester in the active site Asp 20. Also, the article (J.am.chem.Soc.2013, 135, 4580-4583.) reports methods of introducing a photolabile blocking group into the basic C-terminal carboxylate of a PDZ domain ligand to take advantage of synaptic PDZ domain-mediated interactions.
In general, caged peptides are synthesized by standard Solid Phase Peptide Synthesis (SPPS) methods, which provides maximum flexibility in designing caged peptides and protein derivatives. Articles (J.biomed. Mater. Res. PartA 2013,101A, 787-796) produced a photoactivatable peptide containing "RGD" by modifying the carboxylic acid of Asp with 2-nitrobenzyl (2-NB). However, the photochemical and photophysical properties of 2-NB limit its further application in biological processes. 4, 5-Dimethoxynitrobenzyl (DMNB) reported in the article (Peptides 2007,28,1074-1082.) has improved photochemical properties, but it was not incorporated into side chain carboxylic acids during Fmoc SPPS due to the formation of asparagine and pyrrolidone side reactions by Asp (ODMNB) and Glu (ODMNB). The article (Tetrahedron letters, 56 (2015), pp.4582-4585) reports a method for synthesizing a light cage peptide by modification of 4-methoxy-7-nitroindoline (MNI) to derive Asp and Glu. MNI cages have excellent photochemical properties and rapid photolytic kinetics, but the synthesis steps are cumbersome.
Thus, there remains a need for new Fmoc-compatible methods to simply and quickly generate side chain carboxyl blocking peptides/proteins with good photochemical properties.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a glutamic acid derivative compatible with solid phase synthesis and provided with a photosensitive p-methoxybenzoyl group on a side chain, and a synthesis method and application thereof.
The invention firstly carries out substitution reaction on commercially purchased N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester and alpha-bromo-4-methoxyacetophenone, and then removes tert-butyl ester serving as carboxyl protecting group of main glutamic acid chain, thus obtaining the glutamic acid derivative with photosensitive groups on side chains. The glutamic acid derivative disclosed by the invention can be compatible with polypeptide solid-phase synthesis and is used for preparing and producing various polypeptides requiring light-operated protecting groups.
The glutamic acid derivative is abbreviated as Fmoc-Glu (cage) -OH, and has the structural formula shown as follows:
The invention firstly carries out substitution reaction on commercially purchased N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester and alpha-bromo-4-methoxyacetophenone, and then removes tert-butyl ester serving as carboxyl protecting group of main glutamic acid chain, thus obtaining the glutamic acid derivative with photosensitive groups on side chains. The synthetic route is as follows:
The invention discloses a synthesis method of glutamic acid derivative Fmoc-Glu (cage) -OH, which specifically comprises the following steps:
Step 1: synthesis of Compound I
The compound I-1- (tert-butyl) 5- (2- (4-methoxyphenyl) -2-oxoethyl) (((9H-fluoren-9-yl) methoxy) carbonyl) -L-glutamic acid was isolated by dissolving commercially available 1-tert-butyl-D-glutamate with alpha-bromo-4-methoxyacetophenone in dry Dichloromethane (DCM) and adding solid particles of potassium carbonate under argon, stirring at 40 ℃ for 24 hours, concentrating in vacuo after completion of the reaction and purifying by column chromatography.
In the step 1, the molar ratio of the N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester, alpha-bromo-4-methoxyacetophenone and potassium carbonate is 1:1.2:1.5.
Step 2: synthesis of target product
Dissolving a compound I in Dichloromethane (DCM), placing in an ice bath, stirring and cooling to 0 ℃, slowly dropwise adding trifluoroacetic acid (TFA) solution into a reaction system, reacting for 1 hour, removing the ice bath, restoring to room temperature, and continuing stirring and reacting for 6 hours; after the reaction, the solvent is removed by vacuum concentration, and the pure target product (S) -2- (((((9H-fluorene-9-yl) methoxy) carbonyl) amino) -5- (2- (4-methoxyphenyl) -2-oxo ethoxy) -5-oxo valeric acid is obtained.
In step2, the molar ratio of compound I to trifluoroacetic acid (TFA) is 1:60.
In step2, the volume ratio of Dichloromethane (DCM) to trifluoroacetic acid (TFA) in the reaction system was 1:1.
The glutamic acid derivative is used as a special protected amino acid, and is connected to polypeptides by a common N-fluorenylmethoxycarbonyl (Fmoc) solid-phase polypeptide synthesis method, so that various polypeptides requiring light-operated protecting groups are obtained.
The reaction process is schematically as follows:
the beneficial effects of the invention are as follows:
the invention discloses a glutamic acid derivative with a photosensitive p-methoxybenzoyl group on a side chain, a synthesis method and application thereof, wherein the light-operated protection glutamic acid can be compatible with Fmoc solid-phase synthesis for chemically synthesizing polypeptide requiring light-operated protection, and the synthesis method has the characteristics of high synthesis efficiency, simple synthesis steps, high product purity and capability of mass preparation.
Drawings
FIG. 1 is a (Compound I) hydrogen spectrum of Compound I-1- (tert-butyl) 5- (2- (4-methoxyphenyl) -2-oxoethyl) (((9H-fluoren-9-yl) methoxy) carbonyl) -L-glutamic acid hydrogen spectrum-CDCl 3.
FIG. 2 is a hydrogen spectrum of the compound (S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5- (2- (4-methoxyphenyl) -2-oxoethoxy) -5-oxopentanoic acid (Fmoc-Glu (cage) -OH).
FIG. 3 is a carbon spectrum of the compound (S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5- (2- (4-methoxyphenyl) -2-oxoethoxy) -5-oxopentanoic acid (Fmoc-Glu (cage) -OH).
FIG. 4 is a high performance liquid chromatogram of linear polypeptide ARKGKIKPKA-NH 2 prior to ligation of Glu (cage) molecules.
FIG. 5 is a mass spectrum of linear polypeptide ARKGKIKPKA prior to ligation of Glu (cage) molecules.
FIG. 6 is a high performance liquid chromatogram of linear polypeptide GLE (cage) ARKGKIKPKA following ligation of Glu (cage) molecules.
FIG. 7 is a mass spectrum of linear polypeptide GLE (cage) ARKGKIKPKA following ligation of Glu (cage) molecules.
FIG. 8 is a high performance liquid chromatogram of a solid crude peptide SKGLE (cage) ARKGKIKPKA with photo-controlled molecules.
FIG. 9 is a mass spectrum of a solid crude peptide SKGLE (cage) ARKGKIKPKA with photo-controlled molecules.
FIG. 10 is a high performance liquid chromatogram of photopheresis peptide SKGLEARKGKIKPKA.
FIG. 11 is a mass spectrum of photoperiod peptide SKGLEARKGKIKPKA.
FIG. 12 is a high performance liquid chromatogram of solid crude peptide AE (cage) FGLKLDRIG with photo-controlled molecules.
FIG. 13 is a mass spectrum of solid crude peptide AE (cage) FGLKLDRIG with photo-controlled molecules.
FIG. 14 is a high performance liquid chromatogram of photopheresis peptide AEFGLKLDRIG.
FIG. 15 is a mass spectrum of photoperiod peptide AEFGLKLDRIG.
FIG. 16 is a high performance liquid color chart comparing the solid crude peptide AE (cage) FGLKLDRIG before and after removal of the photo-control molecule.
Detailed Description
In order that the invention may be readily understood, a further description of the invention will be rendered by reference to specific embodiments that are merely provided to illustrate the features and advantages of the invention and are not to be construed as limiting the invention to the appended claims.
Example 1:
1. Commercially available N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester (850 mg,1.74 mmol) and alpha-bromo-4-methoxyacetophenone (474 mg,2.08 mmol) were dissolved in 10mL of dried Dichloromethane (DCM) and simultaneously potassium carbonate solid particles (312 mg,2.26 mmol) were added, the reaction was stirred at 40℃for 24 hours under argon, after completion of the reaction, the reaction solution was concentrated in vacuo and isolated by column chromatography purification to give compound I-1- (tert-butyl) 5- (2- (4-methoxyphenyl) -2-oxoethyl) (((9H-fluoren-9-yl) methoxy) carbonyl) -L-glutamic acid (897.6 mg,1.57 mmol) in 90% yield.
2. Compound I (897.6 mg,1.57 mmol) was dissolved in 7mL of Dichloromethane (DCM), placed in an ice bath and stirred to cool to 0℃and 7mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction system, after 1 hour of reaction, the ice bath was removed and the temperature was returned to room temperature and stirring was continued for 6 hours. After the reaction was completed, the solvent was removed by vacuum concentration to give the pure target product, (S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5- (2- (4-methoxyphenyl) -2-oxoethoxy) -5-oxopentanoic acid (Fmoc-Glu (cage) -OH) (731 mg,1.42 mmol), in a yield of 90%.
Example 2:
263.15mg (0.1 mmol) of amino resin having a degree of substitution of 0.38mmol/g was weighed, 10ml of N, N-Dimethylformamide (DMF)/dichloromethane solution (DCM) was added to the resin, the resin was swollen, and the volume ratio of the added solution was: DMF: DCM=1:1, swelling time is 10 minutes, and the swelled product is pumped out by using a diaphragm pump as a power source to obtain the swelled resin.
20% Piperidine was added to the resin and reacted for 10 minutes to completely remove the Fmoc protecting group of the amino group on the resin, and then washed three times with DMF, DCM, DMF each to remove the residual piperidine and the detached small molecule protecting group after the reaction.
The first amino acid Fmoc-Ala-OH (124.53 mg,0.4mmol,4 eq.) and the condensing agent 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate (HCTU, 157.20mg,0.38mmol,3.8 eq.) were dissolved in 4ml DMF, activated for 1 min with N, N-diisopropylethylamine (DIEA, 132. Mu.l, 0.8mmol,8 eq.) and added to the above amino resin, and put into a shaker for 30min at ambient temperature; after completion of the reaction, each was washed three times with DMF, DCM, DMF. Fmoc-Ala-OH (124.53 mg,0.4mmol,4 eq.) and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 144.49mg,0.38mmol,3.8 eq.) and N-hydroxy-7-azabenzotriazole (HOAt, 51.60mg,0.38mmol,3.8 eq.) were added and DIEA (132. Mu.l, 0.8mmol,8 eq.) were dissolved in DMF and put into the resin for 30 minutes; after the completion of the reaction, the resin was washed three times with DMF, DCM, DMF portions of each, then a DMF solution containing 20% (volume fraction) piperidine was added to the resin, and after 5 minutes of the reaction, the resin was washed three times with DMF, DCM, DMF portions of each, and then 20% piperidine was added to the resin again to react for 10 minutes to completely remove the Fmoc protecting group of the amino group on the resin, and then washed three times with DMF, DCM, DMF portions of each to remove the residual piperidine and the detached small molecule protecting group, and the resin was washed with DMF, DCM, DMF portions of each. The subsequent amino acid condensation proceeds similarly until all sequences complete solid phase condensation, ultimately obtaining a linear Fmoc-S (tBu) K (Boc) GLE (cage) AR (Pbf) K (Boc) GK (Boc) IK (Boc) PK (Boc) A-NH 2 resin. The Fmoc protecting group at the N-terminal was removed in two steps with a DMF solution containing 20% piperidine and washed three times with DMF, DCM, DMF, DCM portions each followed by removal of the residual DCM solvent from the resin under reduced pressure to give a dry resin.
Example 3:
To the resin obtained in example 2, 10ml of a pre-prepared cleavage reagent (a mixture of trifluoroacetic acid, phenol, water and triisopropylsilane in a volume ratio of trifluoroacetic acid to water to phenol to triisopropylsilane=88:5:5:2) was added, and the reaction was carried out at room temperature for 2.5 hours, the polypeptide chain was cleaved from the resin, and then the filtrate was collected into a centrifuge tube, and the cleavage liquid was concentrated by a nitrogen bubbling method. Finally, after the cleavage liquid was concentrated to 5ml or less, 30ml of glacial ethyl ether was added for sedimentation, and the crude peptide was allowed to settle to the bottom by centrifugation with a low-speed centrifuge (4500 rpm). Removing supernatant, adding glacial ethyl ether again, ultrasonic treating to form coarse peptide suspension, dissolving small molecule impurity into glacial ethyl ether, and centrifuging. After the completion of the two times, the solid precipitate was air-dried in the shade to obtain 150mg of a solid crude peptide (SKGLE (cage) ARKGKIKPKA) with a photo-controlled molecule.
A small amount of crude peptide was dissolved in pure water, and analyzed by reverse phase high performance liquid chromatography (RP-HPLC) after membrane filtration. The analytical gradient was 10% -70% acetonitrile concentration for 30min. And (3) carrying out ESI-MS identification on the main peak after chromatographic analysis to verify the correctness of the solid crude peptide. After verification, the solid crude peptide was isolated and purified (semi-preparative gradient 10% -70% acetonitrile concentration for 30 min) using a C18 semi-preparative column, and the correct product peak solution was collected and lyophilized in a lyophilizer to give a white flocculent SKGLE (cage) ARKGKIKPKA pure peptide product (112 mg).
Example 4:
The purified peptide product SKGLE (cage) ARKGKIKPKA obtained in example 3 was selected as the target short peptide for further light cage-removal experiments. 1mg SKGLE (cage) ARKGKIKPKA of the pure peptide product was dissolved in 200uL of buffer (6 MGn-HCl,200mM NaH 2 PO4, pH=7.0), placed in an ultraviolet cross-box, placed on flat ice and irradiated with 365nm light for 5min. The reaction was monitored by RP-HPLC. The analytical gradient was 10% -70% acetonitrile concentration for 30min. The main peak was subjected to ESI-MS identification after chromatographic analysis to verify the correctness of the product. The result shows that the pure peptide (CagedForm) of SKGLE (cage) ARKGKIKPKA is mostly converted into SKGLEARKGKIKPKA (NATIVEFORM), which proves that the side chain photosensitive protecting group of Fmoc-Glu (cage) -OH molecule can be well removed under 254nm illumination condition, and has good light cage removal efficiency (see figure 10 and figure 11).
Example 5:
312.5mg (0.1 mmol) of chlorine resin with the substitution degree of 0.32mmol/g is weighed, 10ml of N, N-Dimethylformamide (DMF) solution is added into the resin to swell the resin for 20 minutes, and a diaphragm pump is used as a power source to pump out the swelled product to obtain the swelled resin.
The first amino acid Fmoc-Gly-OH (118.92 mg,0.4mmol,4 eq.) was dissolved in 4ml DMF, N-diisopropylethylamine (DIEA, 132. Mu.l, 0.8mmol,8 eq.) was added to the amino resin after 1 min activation, and the mixture was placed in a shaker and shaken at ambient temperature for 12h; after the completion of the reaction, the resin was washed three times with DMF, DCM, DMF portions of each, a DMF solution containing 20% (volume fraction) piperidine was added to the resin, and after 5 minutes of the reaction, the resin was washed three times with DMF, DCM, DMF portions of each in sequence, and 20% piperidine was added to the resin again to react for 10 minutes to completely remove the Fmoc protecting group of the amino group on the resin. After completion of the reaction, the reaction mixture was washed three times with DMF, DCM, DMF a, fmoc-Ile-OH (141.36 mg,0.4mmol,4 eq.) and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 144.49mg,0.38mmol,3.8 eq.) and N-hydroxy-7-azabenzotriazole (HOAt, 51.60mg,0.38mmol,3.8 eq.) were added and DIEA (132. Mu.l, 0.8mmol,8 eq.) were dissolved in DMF and reacted for 30 minutes; the latter amino acid condensation proceeds similarly until all sequences complete solid phase condensation, finally obtaining a linear Fmoc-AE (cage) FGLK (Boc) LD (OtBu) R (Pbf) IG-COOH resin. The Fmoc protecting group at the N-terminal was removed in two steps with a DMF solution containing 20% piperidine and washed three times with DMF, DCM, DMF, DCM portions each followed by removal of the residual DCM solvent from the resin under reduced pressure to give a dry resin.
Example 6:
To the resin obtained in example 5, 10ml of a pre-prepared cleavage reagent (a mixture of trifluoroacetic acid, phenol, water and triisopropylsilane in a volume ratio of trifluoroacetic acid to water to phenol to triisopropylsilane=88:5:5:2) was added, and the reaction was carried out at room temperature for 2.5 hours, the polypeptide chain was cleaved from the resin, and then the filtrate was collected into a centrifuge tube, and the cleavage liquid was concentrated by a nitrogen bubbling method. Finally, after the cleavage liquid was concentrated to 5ml or less, 30ml of glacial ethyl ether was added for sedimentation, and the crude peptide was allowed to settle to the bottom by centrifugation with a low-speed centrifuge (4500 rpm). Removing supernatant, adding glacial ethyl ether again, ultrasonic treating to form coarse peptide suspension, dissolving small molecule impurity into glacial ethyl ether, and centrifuging. After the completion of the two times, the solid precipitate was air-dried in the shade to obtain 118mg of a solid crude peptide (AE (cage) FGLKLDRIG) with a photo-controlled molecule.
A small amount of crude peptide was dissolved in pure water, and analyzed by reverse phase high performance liquid chromatography (RP-HPLC) after membrane filtration. The analytical gradient was 10% -70% acetonitrile concentration for 30min. And (3) carrying out ESI-MS identification on the main peak after chromatographic analysis to verify the correctness of the solid crude peptide. After verification, the solid crude peptide was isolated and purified (semi-preparative gradient 10% -70% acetonitrile concentration for 30 min) using a C18 semi-preparative column, and the correct product peak solution was collected and lyophilized in a lyophilizer to give a white flocculent AE (cage) FGLKLDRIG pure peptide product (76 mg).
Example 7:
The AE (cage) FGLKLDRIG pure peptide product obtained in example 6 was selected as the target short peptide for further light cage-removal experiments. 1mgAE (cage) FGLKLDRIG pure peptide product was dissolved in 200uL buffer (6M Gn-HCl,200mMNaH 2 PO4, pH=7.0) and placed in an ultraviolet cross-box on flat ice and irradiated with 365nm light for 5min. The reaction was monitored by RP-HPLC. The analytical gradient was 10% -70% acetonitrile concentration for 30min. The main peak was subjected to ESI-MS identification after chromatographic analysis to verify the correctness of the product. The result shows that AE (cage) FGLKLDRIG pure peptide (CagedForm) is mostly converted into AEFGLKLDRIG (NATIVEFORM), which proves that the side chain photosensitive protecting group of Fmoc-Glu (cage) -OH molecule can be well removed under 254nm illumination condition, and has good light cage removal efficiency (see figure 14, figure 15 and figure 16).
In summary, the invention discloses a glutamic acid derivative compatible with solid phase synthesis and provided with a photosensitive p-methoxybenzoyl group on a side chain, and a synthesis method and application thereof.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and the present invention is not limited thereto by the order of the examples, and any changes or substitutions easily suggested by those skilled in the art within the technical scope of the present invention should be included in the scope of the present invention.

Claims (2)

1. A synthesis method of glutamic acid derivatives is characterized in that:
firstly, carrying out substitution reaction on N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester and alpha-bromo-4-methoxyacetophenone, and then removing tert-butyl ester serving as a carboxyl protecting group of a glutamic acid main chain, thus obtaining a glutamic acid derivative with a photosensitive group on a side chain;
The synthetic route is as follows:
The synthesis method comprises the following steps:
Step 1: synthesis of Compound I
Dissolving N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester and alpha-bromo-4-methoxyacetophenone in dry dichloromethane, adding potassium carbonate solid particles, stirring at 40 ℃ for 24 hours under the protection of argon, concentrating in vacuo after the reaction is completed, and purifying and separating by column chromatography to obtain a compound I, namely 1- (tert-butyl) 5- (2- (4-methoxyphenyl) -2-oxo-ethyl) (((9H-fluoren-9-yl) methoxy) carbonyl) -L-glutamic acid;
Step 2: synthesis of target product
Dissolving a compound I in dichloromethane, placing in an ice bath, stirring and cooling to 0 ℃, slowly dropwise adding a trifluoroacetic acid solution into a reaction system, reacting for 1 hour, removing the ice bath, restoring the room temperature, and continuing stirring and reacting for 6 hours; after the reaction is finished, the solvent is removed by vacuum concentration, and the pure target product (S) -2- (((((9H-fluorene-9-yl) methoxy) carbonyl) amino) -5- (2- (4-methoxyphenyl) -2-oxo ethoxy) -5-oxo valeric acid is obtained;
in the step 1, the molar ratio of the N- (9-fluorenylmethoxycarbonyl) -D-glutamic acid 1-tert-butyl ester, alpha-bromo-4-methoxyacetophenone and potassium carbonate is 1:1.2:1.5;
in the step 2, the molar ratio of the compound I to the trifluoroacetic acid is 1:60;
in the step 2, the volume ratio of dichloromethane to trifluoroacetic acid in the reaction system is 1:1.
2. The use of the glutamic acid derivative produced by the synthetic method according to claim 1, characterized in that:
the glutamic acid derivative is connected to the polypeptide by an N-fluorenylmethoxycarbonyl Fmoc solid-phase polypeptide synthesis method, so that the polypeptide requiring the light-operated protecting group is obtained.
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