CN104447759A - Production method for cyclic dipeptide - Google Patents
Production method for cyclic dipeptide Download PDFInfo
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- CN104447759A CN104447759A CN201410686957.6A CN201410686957A CN104447759A CN 104447759 A CN104447759 A CN 104447759A CN 201410686957 A CN201410686957 A CN 201410686957A CN 104447759 A CN104447759 A CN 104447759A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
Abstract
The invention relates to a production method for cyclic dipeptide, and belongs to a natural compound synthesis technology. Cyclic (histidine-proline) dipeptide is prepared by a cooling varying-temperature synthesis and high-pressure high-temperature water phase cyclizing technology. The production method comprises the following steps: taking hydrochloride of histidine proline dipeptide methyl ester as a raw material, and synthesizing high-quality and high-purity cyclic histidine-proline dipeptide by a high-pressure high-temperature assisted method in water containing strong basic and weak acidic salt. Compared with a conventional methanol reflux method, the production method is time-saving, efficient, high in product yield and free of racemization phenomenon.
Description
Technical field
The invention belongs to the synthesis technical field of natural compounds, refer in particular to the production method of a kind of ring (His-Pro) dipeptides.
Background technology
Cyclic dipeptides be by two amino-acid residues by the amido linkage of a pair complementation interconnection and one that is formed stable six-membered ring structure.Because it has the effect of contraction of inherent conformation, comparatively straight chain dipeptides is more stable for Cyclic dipeptides, and not easily by protease hydrolysis, the transformation period is in vivo longer than corresponding two peptide molecules.In addition, two peptide bonds in Cyclic dipeptides molecule become hydrogen-bond donor and hydrogen bond receptor, and hydrogen bond is one of major way of drug receptor interaction, and therefore Cyclic dipeptides is a very important pharmacophore in pharmaceutical chemistry.
Ring (His-Pro) dipeptides [Cyclo (His-Pro), CHP] be a kind of endogenous Cyclic dipeptides, by thyrotrophin-releasing hormone through Pyrrolidonecarboxylic acid aminopeptidase be hydrolyzed slough Pyrrolidonecarboxylic acid after cyclisation form, in the tissue being distributed widely in humans and animals and body fluid.CHP can slow down the anesthetic action of ethanol and hypothermia symptom, resist the toxic side effect of some central depressants, and can stimulate metabolism of blood glucose, plays certain control action kou to the glucose level of type II diabetes people.CHP structure is simple, by hemato encephalic barrier, becomes and effectively treats diabetes, takes drugs, is addicted to drink and the short peptide medicament of one of some behavior disease.
Separation and Extraction CHP not only complex steps and inefficiency from natural product, therefore adopts chemical synthesis synthesis CHP to have great importance.The chemical synthesis of current CHP mainly adopts methanol eddy method, by methyl dipeptide reflux synthesis Cyclic dipeptides in lower boiling methyl alcohol of N-end dissociative.Although methanol eddy method can obtain comparatively ideal efficiency of pcr product, the speed of reaction of this method is very slow, and can cause the racemization of various degree, and the less effective of CHP is even disappeared.
Summary of the invention
The invention provides a kind of production method of Cyclic dipeptides, very slow to solve the speed of reaction existed in current methanol eddy method, and the racemization of various degree can be caused, make the problem that the less effective of CHP even disappears.
The technical scheme that the present invention takes comprises the following steps:
(1) low-temperature protection amino amino
Take L-Histidine as raw material, by its water-soluble-tetrahydrofuran (THF) (v/v=1:2) solvent, the concentration of L-Histidine is 0.25 ~ 0.50mol/L, with tert-Butyl dicarbonate [(Boc)
2o] for protective material carries out amido protecting, first prepare tetrahydrofuran (THF)-(Boc)
2o solution, wherein (Boc)
2o concentration is 1 ~ 1.5mol/L, is added in the alkali aqueous solution containing pH8 ~ 9 of L-Histidine, His ︰ (Boc)
2o (mol/mol)=1:(2 ~ 2.5), stirring reaction 15 ~ 30min at-5 DEG C ~-10 DEG C, stir speed (S.S.) 80 ~ 100r/min, then 30 DEG C ~ 40 DEG C stirring reaction 4 ~ 6h are warming up to, after reaction terminates in temperature 38 DEG C, vacuum tightness be removed under reduced pressure organic solvent under-0.1MPa condition, residuum 1:(18 ~ 20) (g/mL) ratio is dissolved in water, with washed with diethylether 2 ~ 3 times, aqueous phase during each washing: ether phase (v/v)=2:1, removes residual (Boc)
2o, then under 0 DEG C of condition with mass concentration be the citric acid solution acid adjustment of 50% to pH3 ~ 4, obtain the N dissociated
im-Boc-N
α-Boc-L-Histidine; finally be extracted with ethyl acetate 3 ~ 4 times; aqueous phase during each extraction/ethyl acetate phase (v/v)=1 ︰ 1; the amino protected rear L-Histidine of extraction; and then evaporated under reduced pressure removes ethyl acetate under temperature 35 DEG C, vacuum tightness are-0.1MPa condition; obtain amino protected L-Histidine, this process, by the N-Amino End Group of L-Histidine and tertbutyloxycarbonyl (Boc-) protection of side chain imidazolyl, generates N
im-Boc-N
α-Boc-L-Histidine;
(2) low-temperature protection amino acid carboxyl
L-PROLINE is suspended in anhydrous methanol, L-PROLINE concentration in anhydrous methanol is 0.2 ~ 0.5mol/L, be cooled to 0 DEG C, dry HCl gas is passed in system, aeration-agitation reaction 2 ~ 3h, stir speed (S.S.) is 80 ~ 100r/min, and after reaction terminates, under temperature 35 DEG C, vacuum tightness are-0.1MPa condition, reduction vaporization removes methyl alcohol and excessive HCl, obtains the protected L-PROLINE methyl ester hydrochloride of C-end carboxyl;
(3) peptide bond is formed
With methylene dichloride (DCM) for solvent, the N (im) that will prepare through (), (two)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine (N
im-Boc-N
α-Boc-L-His) be dissolved in DCM, adjustment N
im-Boc-N
α-Boc-L-His concentration in DCM is the solution of 0.03 ~ 0.06mol/L, then add 1-hydroxy benzo triazole HOBt and make mixed solution, N'N-dicyclohexylcarbodiimide DCC DCM is dissolved, be mixed with the solution that DCC concentration is 0.3 ~ 0.5mol/L, then under-5 DEG C ~-10 DEG C conditions, this solution is added drop-wise in above-mentioned mixed solution, stirring reaction 20 ~ 30min, stir speed (S.S.) is 80 ~ 100r/min, set up peptide bond forming reactions system, with etc. quality triethylamine in and proline methyl ester hydrochloride (Pro-OMeHCl), then filter with quantitative paper, filtrate joins in above-mentioned peptide bond forming reactions system, after finally adding a small amount of triethylamine adjustment reaction system pH7 ~ 8, then 35 ~ 40 DEG C are warming up to, stirring reaction 2 ~ 4h is continued with 80 ~ 100r/min speed, reaction substrate proportioning is: (N
im-Boc-N
α-Boc-L-His) ︰ (Pro-OMeHCl) ︰ DCC ︰ HOBt (mol/mol)=1 ︰ (1.4 ~ 2.0) ︰ (1.4 ~ 2.0) ︰ (1.4 ~ 2.0), reaction terminates rear solution quantitative paper and filters, to remove by product N, N '-dicyclohexylurea (DCU) (DCU), then in temperature 26 ~ 30 DEG C, vacuum tightness is concentrating under reduced pressure under-0.1MPa condition, the enriched material acetic acid ethyl dissolution obtained dissolves, Nong Suo Wu ︰ ethyl acetate (g/mL)=1:(25 ~ 30), the remaining DCU of filtering and removing, filtrate uses saturated NaCl solution successively, saturated NaHCO
3solution washing 2 ~ 5 times, the saturated NaCl of each ethyl acetate Xiang ︰ or saturated NaHCO
3solution (v/v)=3 ︰ (1 ~ 2), finally be washed till neutrality with saturated NaCl, remove remaining DCU and HOBt, then purification by silica gel column chromatography is adopted, silicagel column filler is granularity 200 ~ 300 order silica gel, eluent is Shi You Mi ︰ ethyl acetate (v/v)=8 ︰ (2 ~ 3), obtains N (im)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine-L-PROLINE methyl esters (N refined
im-Boc-N
α-Boc-L-His-Pro-OMe),
(4) selectively removing of the protected amino of amino acid and carboxyl
Take N prepared by (three)
im-Boc-N
α-Boc-L-His-Pro-OMe is dissolved in ethyl acetate, adjustment N
im-Boc-N
α-Boc-L-His-Pro-OMe concentration is 0.1 ~ 0.3mol/L, dry HCl gas is passed under 0 DEG C of condition, stirring reaction 30 ~ 45min, stir speed (S.S.) 80 ~ 100r/min, after reaction terminates, under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, desolventizing is steamed in decompression, remove Boc-protecting group, obtain retaining the hydrochloride that C-holds the L-Histidine-L-PROLINE methyl esters of protecting group;
(5) Cyclic dipeptides is prepared in the cyclisation of High Temperature High Pressure aqueous phase
L-Histidine-L-PROLINE methyl ester hydrochloride (L-His-L-Pro-OMe2HCl) is suspended in water, is mixed with the suspension that L-His-L-Pro-OMe2HCl concentration is 0.01 ~ 0.05mol/L, uses NaHCO
3be neutralized to pH6.0, L-Histidine-L-PROLINE methyl esters that release is free, then solution sealed, under temperature 130 DEG C ~ 136 DEG C, pressure 0.17 ~ 0.23MPa condition, carry out cyclization 2.5 ~ 3h.Reaction terminates rear solution quantitative paper and filters, filtrate is concentrating under reduced pressure under temperature 45 ~ 50 DEG C, vacuum tightness are-0.1MPa condition, obtain product and adopt purification by silica gel column chromatography, chromatography column filler is granularity 200 ~ 300 order silica gel, eluent Jia Chun ︰ chloroform (v/v)=1 ︰ (4 ~ 5), finally obtains ring (His-Pro) dipeptides faint yellow solid.
The invention provides a kind of production method of Cyclic dipeptides, mainly adopt high-pressure high-temperature technology to carry out Cyclization CHP to methyl dipeptide hydrochloride in water, simple to operate, easily grasp, save time, product yield is high, product yield is more than 93%.Effectively prevent the formation of by product N-acylurea, suppress racemization reaction, occur without racemization phenomenon.Without the need to adopting the catalyzer such as gac in cyclization process, cyclization take water as medium, safety, avoids adopting inflammable and explosive organic solvent, has no irritating odor, no solvent residue.Adopt cheap condensing agent, reduce production cost.Solve when current CHP synthesizes and use that chemical reagent, Product Safety are low, production process has racemization phenomenon to occur in a large number, product efficacy effect reduction is the problem such as disappearance even.
Accompanying drawing explanation
Fig. 1 is the HPLC color atlas of ring (His-Pro) dipeptides reference substance;
Fig. 2 is the HPLC color atlas of two kinds of ring (His-Pro) dipeptides isomer;
Fig. 3 is the HPLC color atlas of standby ring (His-Pro) dipeptides of methanol eddy legal system
Fig. 4 is the HPLC color atlas of ring (His-Pro) dipeptides that HPHT assists cyclisation legal system standby;
Fig. 5 (a) is the ESI-MS mass spectrum of ring (His-Pro) dipeptides reference substance;
Fig. 5 (b) is the ESI-MS mass spectrum of ring (His-Pro) dipeptides trans-isomer(ide);
Fig. 6 (a) is that in Fig. 3, retention time is the ESI-MS mass spectrum of ring (His-Pro) dipeptides of 11.317min (peak A);
Fig. 6 (b) is that in Fig. 3, retention time is the ESI-MS mass spectrum of ring (His-Pro) dipeptides of 11.990min (peak B);
Fig. 7 is that in Fig. 4, retention time is the ESI-MS mass spectrum of ring (His-Pro) dipeptides of 12.036min.
Embodiment
Embodiment 1
(1) low-temperature protection amino amino
Take L-Histidine as raw material, by its water-soluble-tetrahydrofuran (THF) (v/v=1:2) solvent, the concentration of L-Histidine is 0.25mol/L, with tert-Butyl dicarbonate [(Boc)
2o] for protective material carries out amido protecting, first prepare tetrahydrofuran (THF)-(Boc)
2o solution, wherein (Boc)
2o concentration is 1mol/L, is added in the alkali aqueous solution containing the pH8 of L-Histidine, His ︰ (Boc)
2o (mol/mol)=1:2, stirring reaction 15min at-5 DEG C, stir speed (S.S.) 80r/min, then 30 DEG C of stirring reaction 4h are warming up to, be removed under reduced pressure organic solvent under-0.1MPa condition in temperature 38 DEG C, vacuum tightness after reaction terminates, residuum 1:18 (g/mL) ratio is dissolved in water, with washed with diethylether 2 times, aqueous phase during each washing/ether phase (v/v)=2 ︰ 1, removes residual (Boc)
2o, then under 0 DEG C of condition with mass concentration be the citric acid solution acid adjustment of 50% to pH3, obtain the N dissociated
im-Boc-N
α-Boc-L-Histidine; finally be extracted with ethyl acetate 3 times; aqueous phase during each extraction/ethyl acetate phase (v/v)=1 ︰ 1; the amino protected rear L-Histidine of extraction; and then evaporated under reduced pressure removes ethyl acetate under temperature 35 DEG C, vacuum tightness are-0.1MPa condition; obtain amino protected L-Histidine, this process, by the N-Amino End Group of L-Histidine and tertbutyloxycarbonyl (Boc-) protection of side chain imidazolyl, generates N
im-Boc-N
α-Boc-L-Histidine;
(2) low-temperature protection amino acid carboxyl
Be suspended in by L-PROLINE in anhydrous methanol, L-PROLINE concentration in anhydrous methanol is 0.2mol/L, is cooled to 0 DEG C, passes into dry HCl gas in system, and aeration-agitation reaction 2h, stir speed (S.S.) is 80r/min.After reaction terminates, under temperature 35 DEG C, vacuum tightness are-0.1MPa condition, reduction vaporization removes methyl alcohol and excessive HCl, obtains the protected L-PROLINE methyl ester hydrochloride of C-end carboxyl;
(3) peptide bond is formed
With methylene dichloride (DCM) for solvent, the N (im) that will prepare through (), (two)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine (N
im-Boc-N
α-Boc-L-His) be dissolved in DCM, adjustment N
im-Boc-N
α-Boc-L-His concentration in DCM is the solution of 0.03mol/L, then add 1-hydroxy benzo triazole HOBt and make mixed solution, N'N-dicyclohexylcarbodiimide DCC DCM is dissolved, be mixed with the solution that DCC concentration is 0.3mol/L, then under-5 DEG C of conditions, this solution is added drop-wise in above-mentioned mixed solution, stirring reaction 20min, stir speed (S.S.) is 80r/min, set up peptide bond forming reactions system, with etc. quality triethylamine in and proline methyl ester hydrochloride (Pro-OMeHCl), then filter with quantitative paper, filtrate joins in above-mentioned peptide bond forming reactions system, after finally adding a small amount of triethylamine adjustment reaction system pH7, then 35 DEG C are warming up to, stirring reaction 2h is continued with 80r/min speed, reaction substrate proportioning is: (N
im-Boc-N
α-Boc-L-His) ︰ (Pro-OMeHCl) ︰ DCC ︰ HOBt (mol/mol)=1 ︰ 1.4 ︰ 1.4 ︰ 1.4, reaction terminates rear solution quantitative paper and filters, to remove most of by product N, N '-dicyclohexylurea (DCU) (DCU), then concentrating under reduced pressure under temperature 26 DEG C, vacuum tightness are-0.1MPa condition, the enriched material acetic acid ethyl dissolution obtained dissolves, Nong Suo Wu ︰ ethyl acetate (g/mL)=1:25, the remaining DCU of filtering and removing, filtrate is successively with saturated NaCl solution, saturated NaHCO
3solution washing 2 times, the saturated NaCl of each ethyl acetate Xiang ︰ or saturated NaHCO
3solution (v/v)=3 ︰ 1, finally be washed till neutrality with saturated NaCl, remove remaining DCU and HOBt, then purification by silica gel column chromatography is adopted, silicagel column filler is granularity 200 order silica gel, eluent is Shi You Mi ︰ ethyl acetate (v/v)=8 ︰ 2, obtains N (im)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine-L-PROLINE methyl esters (N refined
im-Boc-N
α-Boc-L-His-Pro-OMe),
(4) selectively removing of the protected amino of amino acid and carboxyl
Take N prepared by (three)
im-Boc-N
α-Boc-L-His-Pro-OMe is dissolved in ethyl acetate, adjustment N
im-Boc-N
α-Boc-L-His-Pro-OMe concentration is 0.1mol/L, dry HCl gas is passed under 0 DEG C of condition, stirring reaction 30min, stir speed (S.S.) 80r/min, after reaction terminates, under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, desolventizing is steamed in decompression, remove Boc-protecting group, obtain retaining the hydrochloride that C-holds the L-Histidine-L-PROLINE methyl esters of protecting group;
(5) Cyclic dipeptides is prepared in the cyclisation of High Temperature High Pressure aqueous phase
L-Histidine-L-PROLINE methyl ester hydrochloride (L-His-L-Pro-OMe2HCl) is suspended in water, is mixed with the suspension that L-His-L-Pro-OMe2HCl concentration is 0.01mol/L, uses NaHCO
3be neutralized to pH6.0, L-Histidine-L-PROLINE methyl esters that release is free, then solution is sealed, cyclization 2.5h is carried out under temperature 130 DEG C, pressure 0.17MPa condition, reaction terminates rear solution quantitative paper and filters, filtrate is concentrating under reduced pressure under temperature 45 C, vacuum tightness are-0.1MPa condition, obtain product and adopt purification by silica gel column chromatography, chromatography column filler is granularity 200 order silica gel, eluent Jia Chun ︰ chloroform (v/v)=1 ︰ 4, finally obtains ring (His-Pro) dipeptides faint yellow solid.
Embodiment 2
(1) low-temperature protection amino amino
Take L-Histidine as raw material, by its water-soluble-tetrahydrofuran (THF) (v/v=1:2) solvent, the concentration of L-Histidine is 0.50mol/L, with tert-Butyl dicarbonate [(Boc)
2o] for protective material carries out amido protecting, first prepare tetrahydrofuran (THF)-(Boc)
2o solution, wherein (Boc)
2o concentration is 1.5mol/L, is added in the alkali aqueous solution containing the pH9 of L-Histidine, His ︰ (Boc)
2o (mol/mol)=1:2.5, stirring reaction 30min at-10 DEG C, stir speed (S.S.) 100r/min, then 40 DEG C of stirring reaction 6h are warming up to, be removed under reduced pressure organic solvent under-0.1MPa condition in temperature 38 DEG C, vacuum tightness after reaction terminates, residuum 1:20 (g/mL) ratio is dissolved in water, with washed with diethylether 3 times, aqueous phase during each washing/ether phase (v/v)=2 ︰ 1, removes residual (Boc)
2o, then under 0 DEG C of condition with mass concentration be the citric acid solution acid adjustment of 50% to pH4, obtain the N dissociated
im-Boc-N
α-Boc-L-Histidine; finally be extracted with ethyl acetate 4 times; aqueous phase during each extraction/ethyl acetate phase (v/v)=1 ︰ 1; the amino protected rear L-Histidine of extraction; and then evaporated under reduced pressure removes ethyl acetate under temperature 35 DEG C, vacuum tightness are-0.1MPa condition; obtain amino protected L-Histidine, this process, by the N-Amino End Group of L-Histidine and tertbutyloxycarbonyl (Boc-) protection of side chain imidazolyl, generates N
im-Boc-N
α-Boc-L-Histidine;
(2) low-temperature protection amino acid carboxyl
Be suspended in by L-PROLINE in anhydrous methanol, L-PROLINE concentration in anhydrous methanol is 0.5mol/L, is cooled to 0 DEG C, passes into dry HCl gas in system, and aeration-agitation reaction 3h, stir speed (S.S.) is 100r/min.After reaction terminates, under temperature 35 DEG C, vacuum tightness are-0.1MPa condition, reduction vaporization removes methyl alcohol and excessive HCl, obtains the protected L-PROLINE methyl ester hydrochloride of C-end carboxyl;
(3) peptide bond is formed
With methylene dichloride (DCM) for solvent, the N (im) that will prepare through (), (two)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine (N
im-Boc-N
α-Boc-L-His) be dissolved in DCM, adjustment N
im-Boc-N
α-Boc-L-His concentration in DCM is the solution of 0.06mol/L, then add 1-hydroxy benzo triazole HOBt and make mixed solution, N'N-dicyclohexylcarbodiimide DCC DCM is dissolved, then under-10 DEG C of conditions, this solution is added drop-wise in above-mentioned mixed solution, stirring reaction 30min, stir speed (S.S.) is 100r/min, sets up peptide bond forming reactions system.With etc. quality triethylamine in and proline methyl ester hydrochloride (Pro-OMeHCl), then filter with quantitative paper, filtrate joins in above-mentioned peptide bond forming reactions system, after finally adding a small amount of triethylamine adjustment reaction system pH8, then 40 DEG C are warming up to, continue stirring reaction 4h with 100r/min speed, reaction substrate proportioning is: (N
im-Boc-N
α-Boc-L-His) ︰ (Pro-OMeHCl) ︰ DCC ︰ HOBt (mol/mol)=1 ︰ 2.0 ︰ 2.0 ︰ 2.0, reaction terminates rear solution quantitative paper and filters, to remove most of by product N, N '-dicyclohexylurea (DCU) (DCU), then concentrating under reduced pressure under temperature 30 DEG C, vacuum tightness are-0.1MPa condition, the enriched material acetic acid ethyl dissolution obtained dissolves, Nong Suo Wu ︰ ethyl acetate (g/mL)=1:30, the remaining DCU of filtering and removing, filtrate is successively with saturated NaCl solution, saturated NaHCO
3solution washing 5 times, the saturated NaCl of each ethyl acetate Xiang ︰ or saturated NaHCO
3solution (v/v)=3 ︰ 2, finally be washed till neutrality with saturated NaCl, remove remaining DCU and HOBt, then purification by silica gel column chromatography is adopted, silicagel column filler is granularity 300 order silica gel, eluent is Shi You Mi ︰ ethyl acetate (v/v)=8 ︰ 3, obtains N (im)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine-L-PROLINE methyl esters (N refined
im-Boc-N
α-Boc-L-His-Pro-OMe);
(4) selectively removing of the protected amino of amino acid and carboxyl
Take N prepared by (three)
im-Boc-N
α-Boc-L-His-Pro-OMe is dissolved in ethyl acetate, adjustment N
im-Boc-N
α-Boc-L-His-Pro-OMe concentration is 0.3mol/L, dry HCl gas is passed under 0 DEG C of condition, stirring reaction 45min, stir speed (S.S.) 100r/min, after reaction terminates, under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, desolventizing is steamed in decompression, remove Boc-protecting group, obtain retaining the hydrochloride that C-holds the L-Histidine-L-PROLINE methyl esters of protecting group;
(5) Cyclic dipeptides is prepared in the cyclisation of High Temperature High Pressure aqueous phase
L-Histidine-L-PROLINE methyl ester hydrochloride (L-His-L-Pro-OMe2HCl) is suspended in water, is mixed with the suspension that L-His-L-Pro-OMe2HCl concentration is 0.05mol/L, uses NaHCO
3be neutralized to pH6.0, L-Histidine-L-PROLINE methyl esters that release is free, then solution is sealed, cyclization 3h is carried out under temperature 136 DEG C, pressure 0.23MPa condition, reaction terminates rear solution quantitative paper and filters, filtrate is concentrating under reduced pressure under temperature 50 C, vacuum tightness are-0.1MPa condition, obtain product and adopt purification by silica gel column chromatography, chromatography column filler is granularity 300 order silica gel, eluent Jia Chun ︰ chloroform (v/v)=1 ︰ 5, finally obtains ring (His-Pro) dipeptides faint yellow solid.
Embodiment 3
(1) low-temperature protection amino amino
Take L-Histidine as raw material, by its water-soluble-tetrahydrofuran (THF) (v/v=1:2) solvent, the concentration of L-Histidine is 0.38mol/L, with tert-Butyl dicarbonate [(Boc)
2o] for protective material carries out amido protecting, first prepare tetrahydrofuran (THF)-(Boc)
2o solution, wherein (Boc)
2o concentration is 1.25mol/L, is added in the alkali aqueous solution containing the pH8.5 of L-Histidine, His ︰ (Boc)
2o (mol/mol)=1:2.2.Stirring reaction 25min at-8 DEG C, stir speed (S.S.) 90r/min, then 35 DEG C of stirring reaction 5h are warming up to, after reaction terminates in temperature 38 DEG C, vacuum tightness be removed under reduced pressure organic solvent under-0.1MPa condition, residuum 1:19 (g/mL) ratio is dissolved in water, with washed with diethylether 3 times, aqueous phase/ether phase (v/v)=2 ︰ 1 during each washing, removes residual (Boc)
2o, then under 0 DEG C of condition with mass concentration be the citric acid solution acid adjustment of 50% to pH3.5, obtain the N dissociated
im-Boc-N
α-Boc-L-Histidine, is finally extracted with ethyl acetate 4 times, aqueous phase/ethyl acetate phase (v/v)=1 ︰ 1 during each extraction.The amino protected rear L-Histidine of extraction, and then under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, evaporated under reduced pressure removes ethyl acetate, obtains amino protected L-Histidine.This process, by the N-Amino End Group of L-Histidine and tertbutyloxycarbonyl (Boc-) protection of side chain imidazolyl, generates N
im-Boc-N
α-Boc-L-Histidine;
(2) low-temperature protection amino acid carboxyl
Be suspended in by L-PROLINE in anhydrous methanol, L-PROLINE concentration in anhydrous methanol is 0.35mol/L, is cooled to 0 DEG C, passes into dry HCl gas in system, and aeration-agitation reaction 2.5h, stir speed (S.S.) is 90r/min.After reaction terminates, under temperature 35 DEG C, vacuum tightness are-0.1MPa condition, reduction vaporization removes methyl alcohol and excessive HCl, obtains the protected L-PROLINE methyl ester hydrochloride of C-end carboxyl;
(3) peptide bond is formed
With methylene dichloride (DCM) for solvent, the N (im) that will prepare through (), (two)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine (N
im-Boc-N
α-Boc-L-His) be dissolved in DCM, adjustment N
im-Boc-N
α-Boc-L-His concentration in DCM is the solution of 0.05mol/L, then add 1-hydroxy benzo triazole HOBt and make mixed solution, N'N-dicyclohexylcarbodiimide DCC DCM is dissolved, then under-8 DEG C of conditions, this solution is added drop-wise in above-mentioned mixed solution, stirring reaction 25min, stir speed (S.S.) is 90r/min, set up peptide bond forming reactions system, with etc. quality triethylamine in and proline methyl ester hydrochloride (Pro-OMeHCl), then filter with quantitative paper, filtrate joins in above-mentioned peptide bond forming reactions system, after finally adding a small amount of triethylamine adjustment reaction system pH7.5, then 40 DEG C are warming up to, stirring reaction 3h is continued with 90r/min speed, reaction substrate proportioning is: (N
im-Boc-N
α-Boc-L-His) ︰ (Pro-OMeHCl) ︰ DCC ︰ HOBt (mol/mol)=1 ︰ 1.7 ︰ 1.7 ︰ 1.7, reaction terminates rear solution quantitative paper and filters, to remove most of by product N, N '-dicyclohexylurea (DCU) (DCU), then concentrating under reduced pressure under temperature 28 DEG C, vacuum tightness are-0.1MPa condition, the enriched material acetic acid ethyl dissolution obtained dissolves, Nong Suo Wu ︰ ethyl acetate (g/mL)=1:28, the remaining DCU of filtering and removing, filtrate is successively with saturated NaCl solution, saturated NaHCO
3solution washing 4 times, the saturated NaCl of each ethyl acetate Xiang ︰ or saturated NaHCO
3solution (v/v)=3 ︰ 1.5, finally be washed till neutrality with saturated NaCl, remove remaining DCU and HOBt, then purification by silica gel column chromatography is adopted, silicagel column filler is 300 order silica gel, eluent is Shi You Mi ︰ ethyl acetate (v/v)=8 ︰ 2.5, obtains N (im)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine-L-PROLINE methyl esters (N refined
im-Boc-N
α-Boc-L-His-Pro-OMe),
(4) selectively removing of the protected amino of amino acid and carboxyl
Take N prepared by (three)
im-Boc-N
α-Boc-L-His-Pro-OMe is dissolved in ethyl acetate, adjustment N
im-Boc-N
α-Boc-L-His-Pro-OMe concentration is 0.2mol/L, dry HCl gas is passed under 0 DEG C of condition, stirring reaction 38min, stir speed (S.S.) 90r/min, after reaction terminates, under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, desolventizing is steamed in decompression, remove Boc-protecting group, obtain retaining the hydrochloride that C-holds the L-Histidine-L-PROLINE methyl esters of protecting group;
(5) Cyclic dipeptides is prepared in the cyclisation of High Temperature High Pressure aqueous phase
L-Histidine-L-PROLINE methyl ester hydrochloride (L-His-L-Pro-OMe2HCl) is suspended in water, is mixed with the suspension that L-His-L-Pro-OMe2HCl concentration is 0.03mol/L, uses NaHCO
3be neutralized to pH6.0, L-Histidine-L-PROLINE methyl esters that release is free, then solution is sealed, cyclization 2.7h is carried out under temperature 133 DEG C, pressure 0.2MPa condition, reaction terminates rear solution quantitative paper and filters, filtrate is concentrating under reduced pressure under temperature 48 DEG C, vacuum tightness are-0.1MPa condition, obtain product and adopt purification by silica gel column chromatography, chromatography column filler is granularity 200 order silica gel, eluent Jia Chun ︰ chloroform (v/v)=1 ︰ 4.5, finally obtains ring (His-Pro) dipeptides faint yellow solid.
Following employing HPLC and ESI-MS method carries out analysis contrast to methanol eddy method with by the invention provides the standby Cyclic dipeptides of cyclisation legal system.
By the invention provides the HPLC color atlas of the standby CHP of cyclisation legal system as shown in Figure 4, only there is a peak at 12.036min place in this color atlas, shows that product is optically pure one matter, and mass spectrum corresponding to this peak as shown in Figure 7.The retention time of this Cyclic dipeptides is consistent with mass spectrum and reference substance, can infer thus and the invention provides in method cyclization process the cis structure only defining cyclo (His-Pro).
Table 1 different cyclization method racemization situation contrasts
| Extracting method | Solvent | Time (h) | Yield a(％) | Racemization b(％) |
| Methanol eddy method | Methyl alcohol | 32 | 81 | 31.2 |
| Cyclisation method provided by the invention | Water | 2.5 | 93 | Do not detect |
Note: each reaction repeats 3 times, the yield difference obtained is not higher than ± 5%.
Separation yield after a column chromatography purification, comprises cis-and trans-configuration.
The mensuration of b racemization adopts HPLC method, HPAQ C18 chromatographic column, elutriant acetonitrile/water (2:98).
From the hydrochloride of L-His-L-Pro-OMe, adopt traditional methanol eddy method and this patent to provide cyclization process to synthesize CHP respectively, Cyclic dipeptides yield and the racemization degree of two kinds of cyclization methods are as shown in table 1.Methanol eddy method synthesis rate is slow especially, needs reflux 32h.TLC detects and finds have by product to generate in reaction process, and causes CHP to there occurs the racemization of 31.2%; And adopting the yield of cyclisation method provided by the invention synthesis Cyclic dipeptides higher than methanol eddy method by 15%, the reaction times shortens 10 times; The generation of by product do not detected in reaction process, can not cause racemization yet, product does not detect racemization phenomenon.
Claims (1)
1. a production method for Cyclic dipeptides, is characterized in that comprising the following steps:
(1) low-temperature protection amino amino
Take L-Histidine as raw material, by its water-soluble-tetrahydrofuran (THF) (v/v=1:2) solvent, the concentration of L-Histidine is 0.25 ~ 0.50mol/L, with tert-Butyl dicarbonate [(Boc)
2o] for protective material carries out amido protecting, first prepare tetrahydrofuran (THF)-(Boc)
2o solution, wherein (Boc)
2o concentration is 1 ~ 1.5mol/L, is added in the alkali aqueous solution containing pH8 ~ 9 of L-Histidine, His ︰ (Boc)
2o (mol/mol)=1:(2 ~ 2.5), stirring reaction 15 ~ 30min at-5 DEG C ~-10 DEG C, stir speed (S.S.) 80 ~ 100r/min, then 30 DEG C ~ 40 DEG C stirring reaction 4 ~ 6h are warming up to, after reaction terminates in temperature 38 DEG C, vacuum tightness be removed under reduced pressure organic solvent under-0.1MPa condition, residuum 1:(18 ~ 20) (g/mL) ratio is dissolved in water, with washed with diethylether 2 ~ 3 times, aqueous phase during each washing: ether phase (v/v)=2:1, removes residual (Boc)
2o, then under 0 DEG C of condition with mass concentration be the citric acid solution acid adjustment of 50% to pH3 ~ 4, obtain the N dissociated
im-Boc-N
α-Boc-L-Histidine; finally be extracted with ethyl acetate 3 ~ 4 times; aqueous phase during each extraction/ethyl acetate phase (v/v)=1 ︰ 1; the amino protected rear L-Histidine of extraction; and then evaporated under reduced pressure removes ethyl acetate under temperature 35 DEG C, vacuum tightness are-0.1MPa condition; obtain amino protected L-Histidine, this process, by the N-Amino End Group of L-Histidine and tertbutyloxycarbonyl (Boc-) protection of side chain imidazolyl, generates N
im-Boc-N
α-Boc-L-Histidine;
(2) low-temperature protection amino acid carboxyl
L-PROLINE is suspended in anhydrous methanol, L-PROLINE concentration in anhydrous methanol is 0.2 ~ 0.5mol/L, be cooled to 0 DEG C, dry HCl gas is passed in system, aeration-agitation reaction 2 ~ 3h, stir speed (S.S.) is 80 ~ 100r/min, and after reaction terminates, under temperature 35 DEG C, vacuum tightness are-0.1MPa condition, reduction vaporization removes methyl alcohol and excessive HCl, obtains the protected L-PROLINE methyl ester hydrochloride of C-end carboxyl;
(3) peptide bond is formed
With methylene dichloride (DCM) for solvent, the N (im) that will prepare through (), (two)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine (N
im-Boc-N
α-Boc-L-His) be dissolved in DCM, adjustment N
im-Boc-N
α-Boc-L-His concentration in DCM is the solution of 0.03 ~ 0.06mol/L, then add 1-hydroxy benzo triazole HOBt and make mixed solution, N'N-dicyclohexylcarbodiimide DCC DCM is dissolved, be mixed with the solution that DCC concentration is 0.3 ~ 0.5mol/L, then under-5 DEG C ~-10 DEG C conditions, this solution is added drop-wise in above-mentioned mixed solution, stirring reaction 20 ~ 30min, stir speed (S.S.) is 80 ~ 100r/min, set up peptide bond forming reactions system, with etc. quality triethylamine in and proline methyl ester hydrochloride (Pro-OMeHCl), then filter with quantitative paper, filtrate joins in above-mentioned peptide bond forming reactions system, after finally adding a small amount of triethylamine adjustment reaction system pH7 ~ 8, then 35 ~ 40 DEG C are warming up to, stirring reaction 2 ~ 4h is continued with 80 ~ 100r/min speed, reaction substrate proportioning is: (N
im-Boc-N
α-Boc-L-His) ︰ (Pro-OMeHCl) ︰ DCC ︰ HOBt (mol/mol)=1 ︰ (1.4 ~ 2.0) ︰ (1.4 ~ 2.0) ︰ (1.4 ~ 2.0), reaction terminates rear solution quantitative paper and filters, to remove by product N, N '-dicyclohexylurea (DCU) (DCU), then in temperature 26 ~ 30 DEG C, vacuum tightness is concentrating under reduced pressure under-0.1MPa condition, the enriched material acetic acid ethyl dissolution obtained dissolves, Nong Suo Wu ︰ ethyl acetate (g/mL)=1:(25 ~ 30), the remaining DCU of filtering and removing, filtrate uses saturated NaCl solution successively, saturated NaHCO
3solution washing 2 ~ 5 times, the saturated NaCl of each ethyl acetate Xiang ︰ or saturated NaHCO
3solution (v/v)=3 ︰ (1 ~ 2), finally be washed till neutrality with saturated NaCl, remove remaining DCU and HOBt, then purification by silica gel column chromatography is adopted, silicagel column filler is granularity 200 ~ 300 order silica gel, eluent is Shi You Mi ︰ ethyl acetate (v/v)=8 ︰ (2 ~ 3), obtains N (im)-tertbutyloxycarbonyl-N (α)-tertbutyloxycarbonyl-L-Histidine-L-PROLINE methyl esters (N refined
im-Boc-N
α-Boc-L-His-Pro-OMe),
(4) selectively removing of the protected amino of amino acid and carboxyl
Take N prepared by (three)
im-Boc-N
α-Boc-L-His-Pro-OMe is dissolved in ethyl acetate, adjustment N
im-Boc-N
α-Boc-L-His-Pro-OMe concentration is 0.1 ~ 0.3mol/L, dry HCl gas is passed under 0 DEG C of condition, stirring reaction 30 ~ 45min, stir speed (S.S.) 80 ~ 100r/min, after reaction terminates, under temperature 35 DEG C, vacuum tightness are for-0.1MPa condition, desolventizing is steamed in decompression, remove Boc-protecting group, obtain retaining the hydrochloride that C-holds the L-Histidine-L-PROLINE methyl esters of protecting group;
(5) Cyclic dipeptides is prepared in the cyclisation of High Temperature High Pressure aqueous phase
L-Histidine-L-PROLINE methyl ester hydrochloride (L-His-L-Pro-OMe2HCl) is suspended in water, is mixed with the suspension that L-His-L-Pro-OMe2HCl concentration is 0.01 ~ 0.05mol/L, uses NaHCO
3be neutralized to pH6.0, L-Histidine-L-PROLINE methyl esters that release is free, then solution sealed, under temperature 130 DEG C ~ 136 DEG C, pressure 0.17 ~ 0.23MPa condition, carry out cyclization 2.5 ~ 3h.Reaction terminates rear solution quantitative paper and filters, filtrate is concentrating under reduced pressure under temperature 45 ~ 50 DEG C, vacuum tightness are-0.1MPa condition, obtain product and adopt purification by silica gel column chromatography, chromatography column filler is granularity 200 ~ 300 order silica gel, eluent Jia Chun ︰ chloroform (v/v)=1 ︰ (4 ~ 5), finally obtains ring (His-Pro) dipeptides faint yellow solid.
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| CN106083684A (en) * | 2015-07-27 | 2016-11-09 | 马鞍山德鸿生物技术有限公司 | The preparation method of proline esters hydrochlorate |
| CN106674230A (en) * | 2017-01-04 | 2017-05-17 | 陕西慧康生物科技有限责任公司 | Synthesis method of histidine and proline cyclodipeptide |
| CN109824755A (en) * | 2019-04-09 | 2019-05-31 | 湖南华腾制药有限公司 | N- tertbutyloxycarbonyl-L- leucyl-L-phenylalanine methyl esters production method |
| CN111875668A (en) * | 2020-07-29 | 2020-11-03 | 陕西慧康生物科技有限责任公司 | Synthetic method of cyclic dipeptide containing glutamine and asparagine |
| CN113372278A (en) * | 2021-06-09 | 2021-09-10 | 吉尔多肽生物制药(大连市)有限公司 | Synthesis method of Nalpha-tert-butyloxycarbonyl-Nim-p-toluenesulfonyl-L-histidine |
| CN113861061A (en) * | 2021-10-25 | 2021-12-31 | 成都市科隆化学品有限公司 | Amino acid amide hydrochloride without inorganic ammonium salt and synthetic method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106083684A (en) * | 2015-07-27 | 2016-11-09 | 马鞍山德鸿生物技术有限公司 | The preparation method of proline esters hydrochlorate |
| CN106083684B (en) * | 2015-07-27 | 2018-08-14 | 马鞍山德鸿生物技术有限公司 | The preparation method of proline esters hydrochloride |
| CN106674230A (en) * | 2017-01-04 | 2017-05-17 | 陕西慧康生物科技有限责任公司 | Synthesis method of histidine and proline cyclodipeptide |
| WO2018126523A1 (en) * | 2017-01-04 | 2018-07-12 | 陕西慧康生物科技有限责任公司 | Synthesis method of proline-histidine cyclodipeptide |
| CN106674230B (en) * | 2017-01-04 | 2019-07-02 | 陕西慧康生物科技有限责任公司 | The synthetic method of pro-his cyclic dipeptide |
| CN109824755A (en) * | 2019-04-09 | 2019-05-31 | 湖南华腾制药有限公司 | N- tertbutyloxycarbonyl-L- leucyl-L-phenylalanine methyl esters production method |
| CN111875668A (en) * | 2020-07-29 | 2020-11-03 | 陕西慧康生物科技有限责任公司 | Synthetic method of cyclic dipeptide containing glutamine and asparagine |
| CN113372278A (en) * | 2021-06-09 | 2021-09-10 | 吉尔多肽生物制药(大连市)有限公司 | Synthesis method of Nalpha-tert-butyloxycarbonyl-Nim-p-toluenesulfonyl-L-histidine |
| CN113861061A (en) * | 2021-10-25 | 2021-12-31 | 成都市科隆化学品有限公司 | Amino acid amide hydrochloride without inorganic ammonium salt and synthetic method thereof |
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