CN113968902B - Leucine-enkephalin analogue, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a leucine-enkephalin analogue, which is characterized in that 5 leucine-enkephalin analogues and two analogue intermediates are obtained by carrying out group modification and connection on a4 th phenylalanine side chain aromatic ring structure, the obtained analogues show stronger analgesic effect than leucine-enkephalin, a reliable technical route and product support are provided for further research of the analogues, and meanwhile, ideas are provided for design of synthetic routes and preparation methods of other small peptides.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a leucine-enkephalin analogue, a preparation method and application thereof.
Background
In recent years, delta-opioid receptors have attracted increasing attention as targets for the treatment of chronic pain due to abuse of mu opioid receptor agonists and serious side effects after prolonged use. Compounds that act on delta opioid receptors show fewer side effects than mu opioid receptor agonists.
Enkephalin (Enkephalin) is an oligopeptide (YGGFX) composed of five amino acid residues and is a peptide synthesized by nerve cells and having morphine-like action, so the name Enkephalin belongs to endogenous opioid peptides. Two natural enkephalins exist in the brain, spinal cord and intestinal tract, which differ only in that the C-terminus is leucine and methionine, respectively, and their precursors (pre-enkephalins) are identical. Leucine (leucine enkephalin, leu) 5 Enkephalin, leu-enkephalin): H-Casein-Gly-Phe-Leu-OH, i.e., H-Tyr-Gly-Gly-Phe-Leu-OH; methionine (methionine enkephalin): H-Casein-Gly-Phe-Met-OH, i.e., H-Tyr-Gly-Gly-Phe-Met-OH. They are all central nervous systemsMorphine-like acting neurotransmitters in the system, like opioids, bind to opioid receptors on the cell surface. Neutrazol-containing neurons are found in the brain and polio. Its main function in the spinal cord and brain is to regulate pain sensation.
Leu 5 Enkephalin (H-Tyr) 1 -Gly 2 -Gly 3 -Phe 4 -Leu 5 -OH) is an endogenous opioid peptide, a classical delta-opioid receptor agonist, with powerful analgesic effects. The existing structure-activity relation research shows that: the hydrophobicity and spatial orientation of the 4 th aromatic ring of Leu enkephalin are important for the affinity of delta opioid receptors, so how to modify the structure of enkephalin to have a favorable effect on its structure and physiological activity is a technical problem that the skilled man is urgent to solve.
Disclosure of Invention
The invention aims to: the invention aims to provide a leucine-enkephalin analogue, which is used for improving the analgesic activity and the affinity with delta opioid receptors by modifying and connecting a4 th phenylalanine side chain aromatic ring structure of Leu enkephalin.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a leucine-enkephalin analogue having the structure:
wherein R' is-NH 2 、-NHCH 2 CO 2 Et or-NHCHRCO 2 Et, R is preferably any one of cyclohexyl, t-butyl, ethyl, furyl and benzyl ethyl.
The invention further provides a preparation method of the leucine-enkephalin analogue.
In particular, when R' is-NH 2 The preparation method is as follows: by Fmoc-Phe (4-NO) 2 ) taking-OH as a raw material, adopting Wang resin as a solid phase carrier, and sequentially adding Fmoc-Leu-OH and Fmoc-Phe (4-NO) according to the sequence composition 2 ) -OH, fmoc-Gly-OH, fmoc-Gly-OH, boc-Tyr (tBu) -OH, after condensation is completed, snCl is added 2 ·2H 2 O is reduced, cracked, purified and freeze-dried to obtain H-Tyr-Gly-Gly-Phe (4-NH) 2 )-Leu-OH。
When R' is-NHCH 2 CO 2 Et, the preparation method is as follows: firstly, stirring and mixing 2-chloro-trityl chloride resin, fmoc-Leu-OH and DIEA under the protection of argon, sealing with methanol, washing and drying to obtain Fmoc-Leu-2-chloro-trityl resin, removing amino end protection, and simultaneously activating the subsequent amino acid Fmoc-Phe (4-NH-CH) 2 COOEt) -OH to react at the carboxyl end, then Fmoc-Gly-OH, boc-Tyr (tBu) -OH are sequentially added for coupling reaction, washing and drying are carried out to obtain peptide resin, the peptide resin is cracked, the reaction solution is collected, and then the reaction solution is sequentially concentrated, reconstituted, vacuum filtered, water washed and dried to obtain H-Tyr-Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OH。
When R' is-NHCHRCO 2 Et, wherein, when R is any one of cyclohexyl, tert-butyl, ethyl, furyl and benzyl ethyl, is prepared as follows:
(1) Preparation of Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) by solid phase Synthesis 2 COOEt) -Leu-OH, cleavage from the resin gives Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OH；
(2) By Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH with tert-butyl trichloroacetimidate (TBTA) to Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OtBu；
(3) By Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OtBu is photochemically reacted with alkyl iodides or iodo substituents and cleaved to yield alkyl substituted leucine-enkephalin analogs.
Wherein in the above step, boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH is prepared by the following method: firstly, stirring and mixing the 2-chloro-trityl chloride resin, fmoc-Leu-OH and DIEA under the protection of argon, sealing with methanol, and then washing and drying to obtain the Fmoc-Leu-2-chloro-trityl resinThe amino-terminal protection is then removed, while the subsequent amino acid Fmoc-Phe (4-NH-CH) in the sequence is activated 2 COOEt) -OH to react at the carboxyl end thereof, then sequentially adding Fmoc-Gly-OH, boc-Tyr (tBu) -OH to carry out coupling reaction, washing and drying to obtain peptide resin, cracking the peptide resin, collecting reaction liquid, sequentially concentrating, reconstructing, vacuum filtering, washing with water and drying to obtain Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OH。
Fmoc-Phe(4-NH-CH 2 The preparation method of COOEt) -OH is as follows:
1) Fmoc-Phe (4-NH) 2 ) adding-OH and chloroform into a reaction bottle for dissolution, then adding trichloroacetimidate, concentrating after the reaction is completed, filtering to obtain precipitate, and obtaining Fmoc-Phe (4-NH) 2 ) -OtBu solids;
2) Fmoc-Phe (4-NH) obtained as described above 2 ) Adding absolute ethyl alcohol and sodium carbonate into the-OtBu solid, adding ethyl bromoacetate during stirring, heating and refluxing for reaction, filtering after the reaction is completed, concentrating and drying the reaction solution, and performing column chromatography to finally obtain Fmoc-Phe (4-NH-CH) 2 COOEt) -OtBu solid;
3) The Fmoc-Phe (4-NH-CH) 2 COOEt) -OtBu solid was added to a reaction flask, 50% TFA/DCM was added, the reaction was completed at room temperature, concentrated to an oil, dissolved in ethyl acetate, extracted with 5% sodium bicarbonate, the organic phase was collected, followed by washing with water, saturated brine, drying over anhydrous sodium sulfate, filtration, and the filtrate was collected, concentrated and dried to give Fmoc-Phe (4-NH-CH) 2 COOEt) -OH solids.
Preferably, boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 In COOEt) -Leu-OtBu, boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) was added to the reaction flask 2 COOEt) -Leu-OH, then adding a mixed solution of DCM, TFE and TBTA for dissolution, wherein the mixed solution is mixed according to the volume ratio of DCM: TFE: TBTA=3:1:7, heating in an oil bath and stirring for reaction, TLC detection, adding ice MTBE to precipitate a reactant after the reaction is completed, then stirring the precipitate at 0 ℃, filtering the precipitate, and drying to obtain dry Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OtBu。
Preferably, step (3) is achieved by the following method: boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) was added to a dry quartz tube 2 COOEt) -Leu-OtBu solid, DABCO (triethylenediamine), air was withdrawn and backfilled with argon, acetonitrile was added by syringe, then the tube was transferred into a uv photoreactor, alkyl iodides or other iodo substituents were based on acetonitrile, and slowly added to the reaction solution by syringe pump, the reaction was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated in vacuo, dried, 95% tfa:5%H 2 O is cracked and HPLC purified, and finally the R group substituted leucine-enkephalin analogue is obtained.
The invention further provides application of the leucine-enkephalin analogue in preparing medicines for easing pain.
The beneficial effects are that: according to the invention, leucine-enkephalin is taken as a molecular model, 5 leucine-enkephalin analogues (A1-A5) and two intermediate analogues (A6 and A7) are obtained by modifying a4 th aromatic ring structure, and the obtained analogues show a stronger analgesic effect than the leucine-enkephalin, so that a reliable technical route and product support are provided for further research of the analogues, and meanwhile, ideas are provided for the design of synthetic routes and preparation methods of other small peptides.
Drawings
FIG. 1 is a molecular structure diagram of 5 leucine-enkephalin analogues (A1-A5) and two intermediate analogues (A6 and A7) according to the invention;
FIG. 2 is a graph of HPLC results for Compound A6;
FIG. 3 is an ESI-TOF mass spectrum of Compound A6;
FIG. 4 shows Fmoc-Phe (4-NH-CH) 2 HPLC results for COOEt) -OH;
FIG. 5 shows Fmoc-Phe (4-NH-CH) 2 ESI-TOF mass spectrum of COOEt) -OH;
FIG. 6 is an HPLC plot of Compound A7;
FIG. 7 is an ESI-TOF mass spectrum of Compound A7;
FIG. 8 is an HPLC plot of chemical A1;
FIG. 9 is an ESI-TOF mass spectrum of chemical A1;
FIG. 10 is an HPLC plot of chemical A2;
FIG. 11 is an ESI-TOF mass spectrum of chemical A2;
FIG. 12 is an HPLC plot of chemical A3;
FIG. 13 is an ESI-TOF mass spectrum of chemical A3;
FIG. 14 is an HPLC plot of chemical A4;
FIG. 15 is an ESI-TOF mass spectrum of chemical A4;
FIG. 16 is an HPLC plot of chemical A5;
FIG. 17 is an ESI-TOF mass spectrum of chemical A5;
FIG. 18 is a graph showing analgesic effect of compounds A1-A7;
FIG. 19 is a graph showing analgesic effect of Compound A2;
fig. 20 and 21 show the effect of naloxone on A2 analgesic effect.
Detailed Description
The present invention will be described in further detail with reference to specific examples. Detailed embodiments and specific operations are given, examples will aid in understanding the present invention, but the scope of the present invention is not limited to the following examples. Unless otherwise specified, the reaction materials in the following examples are all commercially available materials.
EXAMPLE 1 Compound A6 (H-Tyr-Gly-Gly-Phe (4-NH) 2 ) -Leu-OH).
By Fmoc-Phe (4-NO) 2 ) taking-OH as a raw material, adopting Wang resin as a solid phase carrier, and sequentially adding Fmoc-Leu-OH and Fmoc-Phe (4-NO) according to the sequence composition 2 ) -OH, fmoc-Gly-OH, fmoc-Gly-OH, boc-Tyr (tBu) -OH, after condensation is completed, snCl is added 2 ·2H 2 O2M (55 eq), reacting for 6h at room temperature, reacting-NO 2 Reduction to-NH 2 Cracking, purifying and freeze-drying to obtain A6. The molecular formula: c (C) 28 H 38 N 6 O 7 Molecular weight: 570.2802.
HPLC detection and MS characterization are carried out on A6, wherein the HPLC detection conditions are as follows: mobile phase a:90% acetonitrile/H 2 O (containing 1% TFA), mobile phase B: H 2 O (1% TFA); flow rate: 1mL/min, detection wavelength: 220nm, elution method: 10% A-100A, running for 30min; the results are shown in FIG. 2. The mass spectrum was detected by ESI-TOF mass spectrometry, and the results are shown in FIG. 3.
EXAMPLE 2 Compound A7 (H-Tyr-Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH).
A7 is prepared by the following method:
(1) Preparation of Fmoc-Leu-2-chloro-trityl resin: 5g of 2-chloro-trityl chloride resin, 1.03mmol/g (1 eq) of substitution value, the peptide synthesizer, the DCM wash, 1.0eq Fmoc-Leu-OH and 2.5eq DIEA dissolved in DCM, and the mixture was stirred mechanically under argon for 1 hour and blocked with methanol. Washed with DMF, DCM and MeOH and dried in vacuo to constant weight to give Fmoc-Leu-2-chloro-trityl resin. The load of the resin was measured by ultraviolet spectrophotometry and found to be 0.8mmol/g.
(2) The resin, after completion of the above preparation, was swollen with 25mL of DCM for 30min, the solvent was drained, the resin was treated with 20% piperidine/DMF for 5min, the amino terminal protection was removed, DMF washed, fmoc by-products (dibenzofulvene and its piperidine adducts) and residual piperidine were removed, and ninhydrin test was performed.
(3) At the same time activating the subsequent amino acid Fmoc-Phe (4-NH-CH) in the sequence 2 COOEt) -OH to react at its carboxy terminus. Fmoc-Phe (4-NH-CH) 2 COOEt) -OH (2 eq), HOBt (2 eq) and DIEA (2 eq) were dissolved in DMF at room temperature, cooled to 0℃and 2eq HBTU was added and the activated amino acid solution was added to the drained resin and the reaction was stirred mechanically for 1 hour. The completion of the condensation was monitored by a qualitative ninhydrin test, and after the completion of the reaction, the resin was drained and washed with DMF.
Wherein Fmoc-Phe (4-NH-CH) 2 COOEt) -OH is prepared by the following synthetic route:
Fmoc-Phe (4-NH) was added to a 50mL round bottom flask 2 ) -OH 5g (12.4 mmol), 100mL of chloroform was dissolved, followed by dropwise addition of trichloroacetimidate (TBTA) (5 eq), the reaction was carried out at 45℃and TLC monitored for progress. After the reaction is completed, concentrating and removing the reaction solvent to obtain oily liquid, adding MTBE, pulping, filtering to obtain Fmoc-Phe (4-NH) 2 ) OtBu solid.
Next, fmoc-Phe (4-NH) obtained as described above was added 2 ) Absolute ethanol (60 mL), sodium carbonate (1.5 eq) and ethyl bromoacetate (1 eq) were added dropwise during stirring to the OtBu solid, which was heated to reflux for 6h. After the reaction is completed, filtering, concentrating and drying the reaction solution, and performing column chromatography to finally obtain Fmoc-Phe (4-NH-CH) 2 COOEt) -OtBu solid.
The Fmoc-Phe (4-NH-CH) 2 COOEt) -OtBu solid was added to a 50mL round bottom flask, 50% TFA/DCM 20mL was added, reacted at room temperature for 1h, after completion of the reaction, concentrated to an oil, dissolved by adding 30mL ethyl acetate, extracted with 5% sodium bicarbonate, the organic phase was collected, followed by washing with water, saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was collected concentrated, dried to give Fmoc-Phe (4-NH-CH) 2 COOEt) -OH solid, 99.7% purity, yield: 85%.
For Fmoc-Phe (4-NH-CH) 2 COOEt) -OH for HPLC detection and MS characterization, wherein the HPLC detection conditions were: mobile phase a:90% acetonitrile/H 2 O (containing 1% TFA), mobile phase B: H 2 O (1% TFA); flow rate: 1mL/min, detection wavelength: 220nm, elution method: 10% A-100A, running for 30min; the results are shown in FIG. 4. The mass spectrum was detected by ESI-TOF mass spectrometry and the results are shown in FIG. 5.
Fmoc-Gly-OH, boc-Tyr (tBu) -OH were added in sequence at 2eq each and the procedure repeated for subsequent monomers of the peptide fragment. After the last coupling reaction, wash with DMF, DCM and MeOH, dry to constant weight in vacuo to give the peptide resin.
(4) The peptide resin was treated with l% TFA/DCM for about 1 hour, then washed twice with 0.5% TFA/DCM for 5min each, and the peptide cleaved from the resin. The reaction was collected, pyridine (1:1 to TFA volume) was added to neutralize excess TFA, concentrated under vacuum, then reconstituted with DMF while continuing to concentrate to remove residual DCM, water was added to precipitate the product, the slurry was stirred at room temperature for 30min, the solid was collected by vacuum filtration, and washed with water. The product was dried in vacuo to give the fully protected Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH in 92% yield and 90% purity. Cracking to obtainCompound A7. The molecular formula: c (C) 32 H 44 N 6 O 9 Molecular weight: 656.3170. HPLC and MS profiles of compound A7 are shown in fig. 6 and 7, respectively.
Compound HPLC detection conditions: mobile phase a:90% acetonitrile/H 2 O (containing 1% TFA), mobile phase B: H 2 O (1% TFA); flow rate: 1mL/min, detection wavelength: 220nm, elution method: 10% A-100A, running for 30min.
Example 3 Synthesis of Compounds A1-A5.
First by Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH with tert-butyl trichloroacetimidate (TBTA) to Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OtBu. Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) was added to a 100mL round bottom flask 2 COOEt) -Leu-OH (1 eq), adding DCM, TFE and TBTA to dissolve in a volume ratio of DCM: TFE: TBTA=3:1:7 solution, heating in an oil bath at 35 ℃ and stirring, reacting for 1h, detecting by TLC, adding ice MTBE 100mL to precipitate the reaction, stirring at 0 ℃ to precipitate for 1h, filtering the precipitate, and drying to obtain dried Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OtBu in 92.8% yield and 91% purity.
Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) was then added to a dry 10mL quartz tube 2 COOEt) -Leu-OtBu solid (0.1 mmol), DABCO (0.5 mmol,56.0 mg), air was evacuated and backfilled with argon (3 times). Acetonitrile (0.4 mL) was added via syringe. Subsequently, the test tube was transferred into an ultraviolet photoreactor, alkyl iodide (0.3 mmol) was dissolved in acetonitrile (0.9 mL), and slowly added to the reaction solution by a syringe pump, and reacted for 18 hours under ultraviolet irradiation. After 18 hours, the reaction was quenched with water (2.0 mL), extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated in vacuo, dried, 95% tfa:5%H 2 O is cracked and purified by HPLC, and finally products A1-A5 are obtained, and the yields after purification of the products are respectively A1: 21 percent, A2:23%, A3:17%, A4:19%, A5:17%. HPLC and MS spectra of A1-A5 are shown in FIGS. 8-17, respectively.
Compound HPLC detection conditions: mobile phase a:90% acetonitrile/H 2 O (containing 1% TFA), mobile phaseB:H 2 O (containing 1% TFA). Flow rate: 1mL/min, detection wavelength: 220nm, elution method: 10% A-100A, running for 30min.
Wherein:
a1: the molecular formula: c (C) 38 H 54 N 6 O 9 Molecular weight: 738.3952.
a2: the molecular formula: c (C) 36 H 52 N 6 O 9 Molecular weight: 712.3796.
a3: the molecular formula: c (C) 34 H 48 N 6 O 9 Molecular weight: 684.3483.
a4: the molecular formula: c (C) 37 H 52 N 6 O 10 Molecular weight: 740.3745.
a5: the molecular formula: c (C) 40 H 52 N 6 O 9 Molecular weight: 760.3796.
example 4 determination of analgesic effects of leucine-enkephalin analogues.
A Kunming male mouse is selected, and the analgesic effect of the medicine on acute pain is detected by using a photo-thermal tail flick instrument. The experimental environment temperature is controlled at 22+/-1 ℃, and the mice can eat and drink water freely before the experiment. For the measurement, the mice were fixed and disposed 3cm from the tail tip of the mice at a beam of light and heat source. And recording the tail flick time of the mice, namely, tail flick latency. Before administration, 3 basal tail flick latencies (3-5 s) were pre-determined, and mice that were too sensitive or insensitive were discarded at 5min intervals. The pain regulation effect of mice in the photo-thermal tail flick experiment was evaluated by using the maximum possible effect MPE (maximum possible effect) value, and the AUC (0-60) represents the area under the curve of 0-60min, which was used to evaluate the analgesic effect of the drug.
We studied Leu in a mouse thermal tail flick test by intrathecal injection (i.t.) A1-A7 5 -analgesic effect of enkephalin and analogues thereof (A1-A7). As shown in fig. 18, intrathecal injection (i.t.) Leu 5 After 30min of enkephalin (1 nmol), the analgesic effect reached the maximum, the whole duration of analgesic effect was 90min. The analgesic activity of intermediates A6 and A7 at a concentration of 3nmol was comparable to that of enkephalin at a concentration of 1 nmol. Under the same administration mode, the analoguesA1 A3, A4 and A5 produce stronger analgesic effect, and the analgesic time is Leu 5 -2.6 times enkephalin. Analogue A2 showed the strongest analgesic effect (as shown in fig. 19), ED 50 The value is 4.77 (4.04-5.62) pmol, and the analgesic effect lasts for 240min.
The anti-analgesic effect of naloxone on analogue A2 was further investigated. The results as shown in fig. 20 and 21 show: intrathecal injection of naloxone (5 nmol) significantly reduced the analgesic effect induced by analog A2 (30 pmol).
The invention provides a design idea and a method for realizing the technical scheme, and the method and the way are numerous, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and the improvements and modifications should be regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (7)
1. A leucine-enkephalin analogue, characterized in that said leucine-enkephalin analogue has the following structure:
wherein R' is-NHCHRCO 2 Et, wherein R is any one of cyclohexyl, t-butyl, ethyl, furyl and benzyl ethyl.
2. A process for the preparation of a leucine-enkephalin analogue according to claim 1, wherein when R' is-NHCHRCO 2 Et, R is any one of cyclohexyl, tertiary butyl, ethyl, furyl and benzyl ethyl, and the preparation method is as follows:
(1) Preparation of Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) by solid phase Synthesis 2 COOEt) -Leu-OH, cleavage from the resin gives Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OH；
(2) By Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OH with tert-butyl trichloroacetimidate to Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OtBu；
(3) By Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt) -Leu-OtBu is photochemically reacted with alkyl iodides or iodo substituents and cleaved to yield alkyl substituted leucine-enkephalin analogs.
3. The process according to claim 2, wherein in step (1), the following amino acids Fmoc-Phe (4-NH-CH) in the activation sequence are removed by stirring and mixing the mixture with 2-chloro-trityl chloride resin, fmoc-Leu-OH and DIEA under argon protection, blocking with methanol, washing and drying to obtain Fmoc-Leu-2-chloro-trityl resin 2 COOEt) -OH to react at the carboxyl end thereof, then sequentially adding Fmoc-Gly-OH, boc-Tyr (tBu) -OH to carry out coupling reaction, washing and drying to obtain peptide resin, cracking the peptide resin, collecting reaction liquid, sequentially concentrating, reconstructing, vacuum filtering, washing with water and drying to obtain Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OH。
4. A method for the preparation of a leucine-enkephalin analogue according to claim 3, wherein Fmoc-Phe (4-NH-CH) 2 The preparation method of COOEt) -OH is as follows:
1) Fmoc-Phe (4-NH) 2 ) adding-OH and chloroform into a reaction bottle for dissolution, then adding trichloroacetimidate, concentrating after the reaction is completed, filtering to obtain precipitate, and obtaining Fmoc-Phe (4-NH) 2 ) -OtBu solids;
2) Fmoc-Phe (4-NH) obtained as described above 2 ) Adding absolute ethyl alcohol and sodium carbonate into the-OtBu solid, adding ethyl bromoacetate during stirring, heating and refluxing for reaction, filtering after the reaction is completed, concentrating and drying the reaction solution, and performing column chromatography to finally obtain Fmoc-Phe (4-NH-CH) 2 COOEt) -OtBu solid;
3) The Fmoc-Phe (4-NH-C)H 2 COOEt) -OtBu solid was added to a reaction flask, 50% TFA/DCM was added, the reaction was completed at room temperature, concentrated to an oil, dissolved in ethyl acetate, extracted with 5% sodium bicarbonate, the organic phase was collected, followed by washing with water, saturated brine, drying over anhydrous sodium sulfate, filtration, and the filtrate was collected, concentrated and dried to give Fmoc-Phe (4-NH-CH) 2 COOEt) -OH solids.
5. The process according to claim 2, wherein in step (2), boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) is added to the reaction flask 2 COOEt) -Leu-OH, then adding a solution of DCM, TFE and TBTA to dissolve, heating in an oil bath and stirring the reaction, after completion of the reaction, adding ice MTBE to precipitate the reaction, then stirring the precipitate at 0deg.C, filtering the precipitate, drying to give dried Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) 2 COOEt)-Leu-OtBu。
6. The process according to claim 2, wherein Boc-Tyr (tBu) -Gly-Gly-Phe (4-NH-CH) is added to the dry quartz tube in step (3) 2 COOEt) -Leu-OtBu solid, triethylenediamine, air was withdrawn and backfilled with argon, acetonitrile was added via syringe, then the tube was transferred into a uv photoreactor, alkyl iodides or other iodo substitutions were based on acetonitrile, and slowly added to the reaction solution via syringe pump, the reaction was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated in vacuo, dried, 95% tfa:5%H 2 O is cracked and HPLC purified, and finally the R group substituted leucine-enkephalin analogue is obtained.
7. Use of the leucine-enkephalin analogue of claim 1 for the preparation of a medicament for analgesia.
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| US4288432A (en) * | 1979-06-12 | 1981-09-08 | Richter Gedeon Vegyeszeti Gyar Rt. | Novel enkephalin analogs and process for the preparation thereof |
| US4304715A (en) * | 1979-06-08 | 1981-12-08 | Derek Hudson | Enkephalin analogues |
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| CN109232748A (en) * | 2018-09-26 | 2019-01-18 | 哈尔滨工业大学 | The cyclisation hybrid peptide and its synthetic method and application that the enkephalins of multidigit point modification is mutually coupled with neurotensin (8-13) |
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| US4304715A (en) * | 1979-06-08 | 1981-12-08 | Derek Hudson | Enkephalin analogues |
| US4288432A (en) * | 1979-06-12 | 1981-09-08 | Richter Gedeon Vegyeszeti Gyar Rt. | Novel enkephalin analogs and process for the preparation thereof |
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