CN101928741A - Production method for preparing endomorphin-1 in organic medium system by chemical enzyme method - Google Patents
Production method for preparing endomorphin-1 in organic medium system by chemical enzyme method Download PDFInfo
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- CN101928741A CN101928741A CN2010102350868A CN201010235086A CN101928741A CN 101928741 A CN101928741 A CN 101928741A CN 2010102350868 A CN2010102350868 A CN 2010102350868A CN 201010235086 A CN201010235086 A CN 201010235086A CN 101928741 A CN101928741 A CN 101928741A
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 19
- ZEXLJFNSKAHNFH-SYKYGTKKSA-N L-Phenylalaninamide, L-tyrosyl-L-prolyl-L-tryptophyl- Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)C1=CC=C(O)C=C1 ZEXLJFNSKAHNFH-SYKYGTKKSA-N 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 108010015205 endomorphin 1 Proteins 0.000 title abstract description 4
- 239000000126 substance Substances 0.000 title abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 239000003960 organic solvent Substances 0.000 claims abstract description 34
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000004365 Protease Substances 0.000 claims abstract description 22
- 108091005804 Peptidases Proteins 0.000 claims abstract description 20
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 19
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 14
- 238000005406 washing Methods 0.000 claims abstract description 10
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 9
- WXYGVKADAIJGHB-ZDUSSCGKSA-N (2s)-3-(1h-indol-2-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound C1=CC=C2NC(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CC2=C1 WXYGVKADAIJGHB-ZDUSSCGKSA-N 0.000 claims abstract description 6
- 239000012044 organic layer Substances 0.000 claims abstract description 5
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 claims description 50
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 229960005181 morphine Drugs 0.000 claims description 24
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 16
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 claims description 13
- 101710176384 Peptide 1 Proteins 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 238000001291 vacuum drying Methods 0.000 claims description 6
- 230000002051 biphasic effect Effects 0.000 claims description 5
- 230000000903 blocking effect Effects 0.000 claims description 5
- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 claims description 4
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- 235000005979 Citrus limon Nutrition 0.000 claims description 4
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- 150000008064 anhydrides Chemical class 0.000 claims description 3
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- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 3
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims 1
- 229940043232 butyl acetate Drugs 0.000 claims 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims 1
- 230000002209 hydrophobic effect Effects 0.000 claims 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims 1
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 10
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- 238000005903 acid hydrolysis reaction Methods 0.000 abstract 1
- 125000006239 protecting group Chemical group 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 235000019419 proteases Nutrition 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- 238000002390 rotary evaporation Methods 0.000 description 6
- 239000000843 powder Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 229920001661 Chitosan Polymers 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- 239000012046 mixed solvent Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- -1 EM-2 Chemical compound 0.000 description 2
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- XIJHWXXXIMEHKW-LJWNLINESA-N endomorphin-2 Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)C1=CC=C(O)C=C1 XIJHWXXXIMEHKW-LJWNLINESA-N 0.000 description 2
- 238000003810 ethyl acetate extraction Methods 0.000 description 2
- MHYCRLGKOZWVEF-UHFFFAOYSA-N ethyl acetate;hydrate Chemical compound O.CCOC(C)=O MHYCRLGKOZWVEF-UHFFFAOYSA-N 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 2
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 101001122476 Homo sapiens Mu-type opioid receptor Proteins 0.000 description 1
- 102100028647 Mu-type opioid receptor Human genes 0.000 description 1
- 108090000137 Opioid Receptors Proteins 0.000 description 1
- 102000003840 Opioid Receptors Human genes 0.000 description 1
- 239000008896 Opium Substances 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000009833 condensation Methods 0.000 description 1
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- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 108010015198 endomorphin 2 Proteins 0.000 description 1
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- PGXWDLGWMQIXDT-UHFFFAOYSA-N methylsulfinylmethane;hydrate Chemical compound O.CS(C)=O PGXWDLGWMQIXDT-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biochemical enzyme catalysis, and particularly relates to a production method for preparing endomorphin-1 in an organic medium system by a chemical enzyme method. The invention adopts organic solvent resistant protease WQ9-2 to react in 50% DMSO reaction system by Boc-Trp and Phe-NH2Synthesis of the dipeptide Boc-Trp-Phe-NH2Separating out product, and acid hydrolysis to remove Boc protecting group to obtain Trp-Phe-NH2(ii) a Adopts organic solvent resistant protease PT121 to catalyze Boc-Tyr-Pro-OH and Trp-Phe-NH in a water-organic solvent two-phase reaction system2Reaction to generate Boc-Tyr-Pro-Trp-Phe-NH2Extracting the product to the upper organic layer, washing the organic layer with water, and removing the solvent to obtain Boc-Tyr-Pro-Trp-Phe-NH2Then acidolyzed to generate endomorphin-1 (Tyr-Pro-Trp-Phe-NH)2). The invention adopts crude enzyme liquid for high-efficiency catalysis, has high yield and low cost compared with commercial enzyme. The organic solvent system adopted in the reaction process realizes the reaction separation coupling of the substrate and the product, greatly improves the yield and simplifies the separation and purification steps of the product.
Description
Technical field
The invention belongs to biochemical industry enzyme technology field, relate to a kind of in the organic medium system production method of morphine peptide in the enzyme method preparation.
Background technology
Interior morphine peptide (Endomorphin) is a kind of tetrapeptide with analgesic activity, and interior morphine peptide-1 (Endomorphin-1, EM-1, Tyr-Pro-Trp-Phe-NH are arranged
2) and interior morphine peptide-2 (Endomorphin-2, EM-2, Tyr-Pro-Trp-Phe-NH
2) two kinds, by the neurobiologist Zadina (Nature of all blue university of the U.S., 1997,386,499-502) in the ox brain, found in 1997, it is present in animal and human's the central nervous system, is considered to μ type opiate receptor (μ-opiate receptor, the endogenous aglucon of high-affinity MOR), highly selective.Its most significant physiological function is the regulation and control for the pain sensation, can also regulate the generation and the release of gastrointestinal motility, neuro-endocrinology hormone in addition.Interior morphine peptide does not have various side effects (Psychopharmacology (Berlin), 2000,151, the 299-305 of other morphine class materials; Am.J.Respir.Crit.CareMed, 2000,162,994-999), as physical interdependence, resistance, respiration inhibition, gastrointestinal discomfort etc., low toxicity efficient because of it, conformation are simple relatively, and for the exploitation of the research in opium field and new and effective anodyne provides pattern more easily, along with going deep into of research, the commerce of interior morphine peptide and analogue thereof and clinical application will become inevitable.Therefore, the morphine peptide is used for research and produces very important in how simple, efficient, the large-scale acquisition.
The amino acid of interior morphine peptide is formed and structure is shown below:
EM-1：Tyr-Pro-Trp-Phe-NH
2 EM-2：Tyr-Pro-Phe-Phe-NH
2
Synthetic chemical method (the Bioorganic ﹠amp that is mainly of morphine peptide in present stage; Medicinal ChemistryLetters, 2000,10,2755-2758; Bioorganic ﹠amp; Medicinal Chemistry, 2005,13,6713-6717; Bioorganic ﹠amp; Medicinal Chemistry, 2007,15,1694-1702; Chemistry ﹠amp; Biodiversity, 2007,4,458-467), but the synthetic peptide matters complex steps of chemical method, easily racemization.At present, have only the report of the synthetic interior morphine peptide of one piece of enzyme process: 2008, what equality people (Chinese Journal ofBiochemistry ﹠amp; Molecular Biology, 2008,24,426-431) adopt enzyme process to synthesize interior morphine peptide-1, its with acetonitrile as organic medium, in little water organic solvent system with Boc-Trp and Phe-NH
2Be substrate, with the synthetic Trp-Phe-NH of sodium alginate-chitosan immobilized papain (IPSAC) catalysis
2, productive rate is 27.8%.In same reaction system with Boc-Tyr-Pro-OMe and Trp-Phe-NH
2Be substrate, with the synthetic Tyr-Pro-Trp-Phe-NH of sodium alginate-chitosan immobilized papain (IPSAC) catalysis
2, productive rate is 35.2%.
In the system of morphine peptide-1, the sodium alginate-chitosan immobilized papain was expensive, and activity and the less stable of common proteolytic enzyme in the organic phase system, causes productive rate lower in above-mentioned enzyme process was synthetic.And acetonitrile has bigger toxicity as the organic phase reaction system.Derive from the organic solvent tolerant protease of natural organic solvent-resistant microorganism, can stable existence in organic solvent, can be in organic solvent the dehydration condensation of catalysis peptide efficiently.The present invention has adopted the organic solvent tolerant protease crude enzyme liquid that screens voluntarily, and organic solvent tolerance is strong, and is low with respect to the commercial enzyme cost, and the productive rate of morphine peptide-1 reached about 90% in catalysis was synthetic; In addition, in organic solvent tolerant protease catalysis is synthetic in the reaction of morphine peptide-1, water-dimethyl sulfoxide (DMSO) (DMSO) single_phase system, water-ethyl acetate diphasic system have been adopted, in two kinds of systems, all realized the Reaction Separation coupling of substrate and product, improve productive rate greatly, simplified the separation and purification of product.Through inventor's research with keen determination, found a kind of organic solvent tolerant protease morphine peptide-1 synthetic approach in high yield, easy, the green catalysis in organic medium that adopts.
Summary of the invention
The objective of the invention is to propose the technology of the interior morphine peptide of enzyme method preparation in a kind of organic medium system, this technology adopts synthetic dipeptides of organic solvent tolerant protease catalysis and tetrapeptide respectively in water-organic solvent single_phase system and water-organic solvent biphasic system, and realized the Reaction Separation coupling, improve productive rate greatly, be convenient to product and separate.The key step and the reaction formula of this production technique are as follows:
1) adopt mixed anhydride method by Boc-Tyr and the synthetic dipeptides Boc-Tyr-Pro-OMe of Pro-OMe;
2) methanol solution of Boc-Tyr-Pro-OMe adds an amount of 1mol/L NaOH solution reaction, is 3 with the lemon acid for adjusting pH afterwards, obtains Boc-Tyr-Pro-OH;
3) organic solvent tolerant protease WQ9-2 in the 50%DMSO reaction system by Boc-Trp and Phe-NH
2Synthetic dipeptides Boc-Trp-Phe-NH
2, product is directly separated out from reaction system, washing, suction filtration, vacuum-drying;
4) adopt 50% (v/v) trifluoroacetic acid to remove Boc-Trp-Phe-NH
2The Boc blocking group, obtain Trp-Phe-NH
2
5) organic solvent tolerant protease PT121 catalysis Boc-Tyr-Pro-OH and Trp-Phe-NH in water-organic solvent biphasic reaction system
2Reaction generates Boc-Tyr-Pro-Trp-Phe-NH
2, product is extracted to goes up the phase organic layer, gets the upper strata, and washing is revolved to steam to remove and is desolvated vacuum-drying;
6) adopt 50% (v/v) trifluoroacetic acid to remove Boc-Tyr-Pro-Trp-Phe-NH equally
2The Boc blocking group, obtain in morphine peptide-1 (Tyr-Pro-Trp-Phe-NH
2).
Boc-Tyr+Pro-OMe→Boc-Tyr-Pro-OH
Boc-Trp-Phe-NH
2→Trp-Phe-NH
2
Boc-Tyr-Pro-Trp-Phe-NH
2→Tyr-Pro-Trp-Phe-NH
2
Dipeptides Boc-Tyr-Pro-OMe is synthetic by mixed anhydride method, and substrate B oc-Tyr and Pro-OMeHCl mol ratio are about 1: 1, and-15 ℃ were reacted 1.5 hours, and product is a yellow oil, productive rate 95.6%.This yellow oil is dissolved in methyl alcohol, adds 1M NaOH solution, and room temperature reaction 10 hours with 10% (w/v) lemon acid for adjusting pH to 3, is used ethyl acetate extraction, revolves steaming, and getting white solid is Boc-Tyr-Pro-OH, productive rate 87%.
Dipeptides Boc-Trp-Phe-NH
2By coming from Bacillus cereus WQ9-2 (CCTCCM2010010; Application number: organic solvent tolerant protease WQ9-2 201010103804.6) is synthetic in the 50%DMSO reaction system, and reaction conditions is: 100mM Boc-Trp and 200mM Phe-NH
2, 37 ℃, 200rpm reacted 2～5 hours, and product is directly separated out from reaction system, and organic solvent DMSO and residue substrate are removed in washing, suction filtration, vacuum-drying must be than pure products, productive rate 87%～95%.
Tetrapeptide Boc-Tyr-Pro-Trp-Phe-NH
2By coming from Pseudomonas aeruginosa PT121 (CCTCCM208029; Publication number: organic solvent tolerant protease PT121 CN 101240254A) is synthetic in water-ethyl acetate biphasic reaction system, 20mM Boc-Tyr-Pro-OH and 60mM Trp-Phe-NH
2Be dissolved in ethyl acetate, the PT121 crude enzyme liquid of 1/3rd ethyl acetate volumes adds reaction flask, 37 ℃, 250rpm reacted 8 hours, product is extracted to ethyl acetate layer, get the phase ethyl acetate layer, use saturated sodium bicarbonate solution, 10% (w/v) citric acid solution, water, saturated nacl aqueous solution washing successively, rotary evaporation is removed solvent, get yellow powder powder material, productive rate 85%～95%.
Dipeptides Boc-Trp-Phe-NH
2And tetrapeptide Boc-Tyr-Pro-Trp-Phe-NH
2Following method is adopted in the removal of middle Boc blocking group: quantity of sample is dissolved in an amount of methylene dichloride; ice bath adds the trifluoroacetic acid of equivalent down then; room temperature reaction 6 hours; the mixed solvent that adds V (methylene dichloride): V (methyl alcohol)=2: 1; to take away trifluoroacetic acid; rotary evaporation removes and desolvates, and adds the anhydrous diethyl ether product and separates out.
Beneficial effect of the present invention is as follows:
1. adopt the organic solvent tolerant protease that screens voluntarily to carry out catalyzed reaction, well solved enzyme easy inactivation in organic solvent, the problem of poor stability has improved productive rate greatly.It is synthetic to adopt crude enzyme liquid to carry out catalysis, has reduced reaction cost.
2. in selected organic reaction system, product is directly separated out from reaction system or is extracted to organic layer in the biphasic reaction, has well realized separating of reaction substrate and product, has improved yield, and intermediate product needn't separation and purification.
3. research trial of the present invention goes out a kind of effective employing organic solvent tolerant protease morphine peptide-1 synthetic approach in the catalysis in organic medium, the productive rate height, and purifying is simple, and is with low cost, for the commercial scale production of interior morphine peptide provides the reliable technique support.
Embodiment
Embodiment one
The chemosynthesis of Boc-Tyr-Pro-OH
The 100mL reactor adds methylene dichloride 10mL, Boc-Tyr-OH 0.625g, place-15 ℃ cryosel to bathe, add N-methylmorpholine (NMM) 0.5mL, isobutyl chlorocarbonate 0.3mL, leave standstill 5min, add L-Pro-OMeHCl 0.47g, TLC detects to reacting and finishes, evaporated under reduced pressure adds the extraction of an amount of ethyl acetate and water, and organic phase is successively with saturated sodium bicarbonate solution, 10% citric acid solution, water, saturated nacl aqueous solution washing, rotary evaporation is removed solvent, gets light yellow dope Boc-Tyr-Pro-OMe.
0.7g (2.0mmol) Boc-Tyr-Pro-OMe is dissolved in the NaOH aqueous solution that an amount of methyl alcohol adds 10ml1mol/L, room temperature reaction 10-12h adds the saturated NH of 20ml
4Cl solution is 3 with 10% (w/v) lemon acid for adjusting pH, uses ethyl acetate extraction, water, saturated common salt water washing successively, anhydrous MgSO
4Dry evaporated under reduced pressure gets white powder substance B oc-Tyr-Pro-OH.
Embodiment two
Trp-Phe-NH
2Enzyme process synthetic
0.6g (100mM) Boc-Trp, 0.73g (200mM) Phe-NH
2Be dissolved in 10mLDMSO, slowly add the crude enzyme liquid of 10mL organic solvent tolerant protease WQ9-2, shake up, 37 ℃ of constant temperature, 200rpm, the 2h after product is separated out, and washes with water, and is centrifugal, and vacuum-drying gets white powder substance B oc-Trp-Phe-NH
2
0.9g Boc-Trp-Phe-NH
2Be dissolved in the 6ml methylene dichloride, ice bath slowly drips 6ml trifluoroacetic acid (TFA) down, 27～28 ℃ of reaction 6h, rotary evaporation removes and desolvates, the mixed solvent that adds 7.5ml V (methylene dichloride): V (methyl alcohol)=2: 1, solvent removed in vacuo to be taking away residual trifluoroacetic acid (TFA), adds the 20ml anhydrous diethyl ether and leaves standstill that to separate out solid after for some time be Trp-Phe-NH
2
Embodiment three
Tyr-Pro-Trp-Phe-NH
2Enzyme process synthetic
0.042g (20mM) Boc-Tyr-Pro-OH and 0.084g (60mM) Trp-Phe-NH
2Be dissolved in 6ml water saturation ethyl acetate, the crude enzyme liquid that adds 3ml organic solvent tolerant protease PT121,37 ℃, 250rpm reaction 8～10 hours, get the upper strata ethyl acetate, use saturated sodium bicarbonate solution, 10% (w/v) citric acid solution, water, saturated nacl aqueous solution washing successively, rotary evaporation is removed solvent, get buff powder, be Boc-Tyr-Pro-Trp-Phe-NH
2
0.14g (0.1mmol) Boc-Tyr-Pro-Trp-Phe-NH
2Be dissolved in the 3ml methylene dichloride, ice bath slowly drips 3ml trifluoroacetic acid (TFA) down, 27～28 ℃ of reaction 6h, rotary evaporation removes and desolvates, the mixed solvent that adds 6ml V (methylene dichloride): V (methyl alcohol)=2: 1, solvent removed in vacuo to be taking away residual trifluoroacetic acid (TFA), adds the 20ml anhydrous diethyl ether and leaves standstill that to separate out solid after for some time be Tyr-Pro-Trp-Phe-NH
2
Claims (3)
1. production method of morphine peptide-1 in the enzyme method preparation in the organic medium system is characterized by by following steps and constitutes:
1) adopt mixed anhydride method by Boc-Tyr and the synthetic dipeptides Boc-Tyr-Pro-OMe of Pro-OMe;
2) methanol solution of Boc-Tyr-Pro-OMe adds an amount of 1mol/L NaOH solution reaction, is 3 with the lemon acid for adjusting pH afterwards, obtains Boc-Tyr-Pro-OH;
3) organic solvent tolerant protease WQ9-2 in the 50%DMSO reaction system by Boc-Trp and Phe-NH
2Synthetic dipeptides Boc-Trp-Phe-NH
2, product is directly separated out from reaction system, washing, suction filtration, vacuum-drying;
4) adopt 50% (v/v) trifluoroacetic acid to remove Boc-Trp-Phe-NH
2The Boc blocking group, obtain Trp-Phe-NH
2
5) organic solvent tolerant protease PT121 catalysis Boc-Tyr-Pro-OH and Trp-Phe-NH in water-organic solvent biphasic reaction system
2Reaction generates Boc-Tyr-Pro-Trp-Phe-NH
2, product is extracted to goes up the phase organic layer, gets the upper strata, and washing is revolved to steam to remove and is desolvated vacuum-drying;
6) adopt 50% (v/v) trifluoroacetic acid to remove Boc-Tyr-Pro-Trp-Phe-NH equally
2The Boc blocking group, obtain in morphine peptide-1 (Tyr-Pro-Trp-Phe-NH
2).
2. according to the production method of the described synthetic interior morphine peptide-1 of claim 1, it is characterized in that: Boc-Trp-Phe-NH
2The organic solvent tolerant protease WQ9-2 that adopts in synthetic comes from Bacillus cereusWQ9-2 (CCTCCM2010010; Application number: 201010103804.6), hydrophilic organic solvent wherein is dimethyl sulfoxide (DMSO), dimethyl formamide, methyl alcohol, ethanol, acetonitrile, Virahol, glycerol, ethylene glycol, 1,2-propylene glycol, 1, a kind of in the 4-butyleneglycol or their mixture.
3. according to the production method of the described synthetic interior morphine peptide-1 of claim 1, it is characterized in that: Boc-Tyr-Pro-Trp-Phe-NH
2The organic solvent tolerant protease PT121 that adopts in synthetic comes from Pseudomonas aeruginosa PT121 (CCTCCM208029; Publication number: CN 101240254A), hydrophobic organic solvent wherein is a kind of in ethyl acetate, butylacetate, propyl carbinol, isopropylcarbinol, primary isoamyl alcohol, chloroform, the methylene dichloride etc. or their mixture.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242497A (en) * | 2013-05-01 | 2013-08-14 | 吉林大学 | Method for synthesising diblock copolymer by simultaneous chemoenzymatic process and one-pot process |
| CN108101978A (en) * | 2017-12-18 | 2018-06-01 | 哈尔滨工业大学 | The Tyr-Pro-Trp-Phe-NH2 analog and its synthetic method of the esterification modification of C- terminal aromatics and application |
-
2010
- 2010-07-23 CN CN2010102350868A patent/CN101928741A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242497A (en) * | 2013-05-01 | 2013-08-14 | 吉林大学 | Method for synthesising diblock copolymer by simultaneous chemoenzymatic process and one-pot process |
| CN108101978A (en) * | 2017-12-18 | 2018-06-01 | 哈尔滨工业大学 | The Tyr-Pro-Trp-Phe-NH2 analog and its synthetic method of the esterification modification of C- terminal aromatics and application |
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