CN101274958A - Bioactive peptide, preparation thereof and application thereof - Google Patents
Bioactive peptide, preparation thereof and application thereof Download PDFInfo
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- CN101274958A CN101274958A CNA2008100162496A CN200810016249A CN101274958A CN 101274958 A CN101274958 A CN 101274958A CN A2008100162496 A CNA2008100162496 A CN A2008100162496A CN 200810016249 A CN200810016249 A CN 200810016249A CN 101274958 A CN101274958 A CN 101274958A
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Abstract
The invention relates to a biological bioactive peptide, and the molecular formula thereof is C42H67N7O10. When in preparation, tetrapeptide fragment N-(t-butoxycarbonyl)-L-isoleucine-N-methyl-D-phenylalanine-L-proline methyl ester is firstly prepared (t-butoxycarbonyl: BOC); then N-(9-fluorenylmethyloxycarbonyl)-N-methyl-glutamine is prepared (fluorenylmethyloxycarbonyl: Fmoc); then dipeptide fragment L-2-hydroxyl-3-methyl phocenic acid-O-benzyl salicylate-leucine is prepared; finally, the tetrapeptide fragment is detached from protecting groups and goes through condensation with N-(9-Fmoc)-N-methyl-glutamine, thus obtaining pentapeptide fragment N-(9-Fmoc)-N-methyl-glutamine-L-isoleucine-N-methyl-D-phenylalanine-L-proline methyl ester which goes through condensation after the dipeptide fragment and the pentapeptide fragment are detached from protecting groups. The biological bioactive peptide can provide sufficient raw materials and effective synthetic routes for the research of anti-tumor medicines and has a simple and convenient preparation method and easily controllable conditions.
Description
Technical field
The present invention relates to a kind of biologically active peptides and preparation method thereof and in the application of anti-tumor aspect, this biologically active peptides be a kind of from the ocean, have chain type seven peptides of anti-tumor activity.
Background technology
The marine bioactivity polypeptide is at present mostly from sponge, Ascidian, marine alga and sea hare etc., and its novel structure and significant biological activity have caused the very big interest of synthetic chemistry man and Pharmaceutical Chemist.Yet, being rich in N-methylamino acid and other amino acid that highly makes a variation in the active polypeptide of marine source more, this brings great challenge for synthetic chemistry man.
Tasiamide is Moore group (Williams, P.G.; Yoshida, W.Y.; Moore, R.E.; Paul, V.J., J.Nat.Prod., 2002,65,1336-1339) chain type seven peptides of a novel structure that from the blue or green bacterium Symploca of ocean algae sp., is separated to, it is to the IC of KB and LoVo cell
50Value is respectively 0.48 and 3.47 μ g/mL, the three-dimensional absolute configuration that Moore group infers this bioactive peptide at first for (1S, 8R, 20S, 26S, 32S, 38S), structure is as follows:
Yet, along with further investigation to this active substance, the inventor find above-mentioned structure of inferring and incorrect, the correct three-dimensional absolute configuration of this biologically active peptides be (1S, 8R, 20S, 26R, 32S, 38S).Therefore synthetic biologically active peptides with correct structure is very important to the structure activity study of understanding and carry out the bioactive peptide medicine.Because the amount of natural product is very few, purifying, separation, evaluation are all comparatively difficult in addition, have limited the activity research progress of marine bioactivity peptide greatly.Wherein chemosynthesis is exactly a kind of alternative method, therefore develops a kind of effective method for preparing biologically active peptides and is very important.
Summary of the invention
The purpose of this invention is to provide effective preparation method of a kind of biologically active peptides and application, it can provide enough raw material and effective synthetic route for the research of antitumor drug.
A kind of biologically active peptides, the molecular formula that it is characterized in that it is C
42H
67N
7O
10, structural formula is:
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26R, 32S, in the time of 38S), stereostructural formula is
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26S, 32R, in the time of 38S), stereostructural formula is
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26R, 32R, in the time of 38S), stereostructural formula is
The preparation method of above-mentioned biologically active peptides, it is characterized in that at first making the L-proline(Pro) in the presence of thionyl chloride and methyl alcohol, to methylate, obtain the hydrochloride of L-proline methyl ester, making N-tertbutyloxycarbonyl-D-phenylalanine carry out nitrogen under the effect of sodium hydride and methyl iodide methylates, obtain N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine, hydrochloride and the condensation under the effect of the hydrochloride of condensing agent 1-hydroxyl-7-azo benzotriazole and 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide of N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine with above-mentioned L-proline methyl ester, obtain two peptide fragment N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine-L-proline methyl esters, remove the segmental tertbutyloxycarbonyl protecting group of this dipeptides, again with N-(9-fluorenes methoxy carbonyl acyl group)-glycine condensation, obtain tripeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-glycine-N-methyl D-phenylalanine-L-proline methyl ester, remove the 9-fluorenes methoxy carbonyl acyl group protecting group of this tripeptide fragment, with N-(tertbutyloxycarbonyl)-L-Isoleucine condensation, obtain tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester again; Then make N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-glutamine Sheng under the effect of Paraformaldehyde 96 and tosic acid is Chenged the oxazolidone intermediate, this intermediate reductive ring open and remove trityl-protecting group and obtain N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine in the presence of trifluoroacetic acid and triethyl silicane; Make the L-Isoleucine obtain L-2-hydroxy-3-methyl valeric acid then by the method for azo intermediate hydrolysis, leucine is refluxed in the presence of toluene, benzylalcohol and tosic acid obtain O-benzyl ester-leucic tosilate, the condensation under the effect of the hydrochloride of condensing agent 1-hydroxyl-7-azo benzotriazole and 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide with above-mentioned L-2-hydroxy-3-methyl valeric acid and O-benzyl ester-leucine tosilate obtains L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-leucine; The last tertbutyloxycarbonyl protecting group that under the effect of trifluoroacetic acid, removes above-mentioned tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; and with N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine condensation; obtain pentapeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; this pentapeptide fragment removes 9-fluorenes methoxy carbonyl acyl group protecting group; obtain intermediate N methyl-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; above-mentioned L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-leucine after removing benzyl under the hydrogenolysis condition of palladium preparation, is obtained biologically active peptides of the present invention with above-mentioned intermediate condensation again.
The application of above-mentioned biologically active peptides in suppressing the kinds of tumor cells growth.
The invention provides a kind of biologically active peptides and effectively preparation method and application thereof, enough raw material and effective synthetic routes can be provided for the research of antitumor drug, the preparation method is easy, condition is easy to control, and can be drug screening provides structure different compounds with further structure activity study.
Description of drawings
Embodiment
The three-dimensional absolute configuration of biologically active peptides of the present invention be (1S, 8R, 20S, 26R, 32S, in the time of 38S), called after Tasiamide-D-Gln, its preparation method may further comprise the steps:
1. the preparation of tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester, it is respectively as follows step by step:
Step a: make the L-proline(Pro) under the condition of methyl alcohol and thionyl chloride, carry out esterification.During operation 50mL methyl alcohol is cooled to-10 ℃, stirs and slowly splash into 13mL thionyl chloride (SOCl down
2), add 5.75g (50mmol) L-proline(Pro) (L-Pro-OH) behind the 25min, stirred 2 days under the room temperature.Thin-layer chromatography (TLC) removes solvent under reduced pressure after showing that reaction finishes, and adds 25mL methyl alcohol again and concentrates 2 times repeatedly, add the 100mL ether in residue, cryosel is bathed down and is ground suction filtration, get light yellow solid O-methyl-L-proline hydrochlorate (O-Me-L-Pro.HCl) 5.4g, yield 65%.
Step b: 530mg (2mmol) N-tertbutyloxycarbonyl-D-phenylalanine (Boc-D-Phe-OH) is dissolved in the 10mL anhydrous tetrahydro furan (THF), under ice bath, adds 1.02g (7.2mmol) methyl iodide (CH
3I), 2 batches of branches add 256mg (7.2mmol) NaH (concentration expressed in percentage by weight is 60%) altogether after 10 minutes, finish to make it rise to room temperature naturally, and continue to stir to steam behind the 24h and desolventize, resistates is dissolved in the 200mL distilled water, and, uses 20mL at every turn with normal hexane washing three times.After water layer was transferred pH=1 with hydrochloric acid, with ethyl acetate extraction 5 times, the volume with ethyl acetate was 40mL at every turn.Combining extraction liquid is also with 10% (concentration expressed in percentage by weight, sodium thiosulfate solution down together) is given a baby a bath on the third day after its birth inferior, each 20mL, again with after the saturated aqueous common salt 20mL washing once, the ethyl acetate organic layer is concentrated, get N-tertbutyloxycarbonyl-N-methyl D-phenylalanine (Boc-N-Me-D-Phe-OH) 552mg, yield 99%.
Step c: synthetic two peptide fragment N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine-L-proline methyl esters under the effect of condensing agent.During operation, 644.3mg (3.89mmol) O-Me-L-Pro.HCl is dissolved in the 20mL methylene dichloride, ice bath adds 391.4mg (4.66mmol) sodium bicarbonate down, after 10 minutes, hydrochloride (EDC.HCl) 894.4mg (4.66mmol) that adds 1.09g (3.89mmol) Boc-N-Me-D-Phe-OH, condensing agent 1-hydroxyl-7-azo benzotriazole (HOAt) 634.2mg (4.66mmol) and 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide successively, ice bath after 30 minutes room temperature reaction spend the night.After TLC shows that reaction finishes, steaming desolventizes, add 200mL ethyl acetate dilute reaction solution, use 5% sodium bicarbonate aqueous solution, saturated common salt water washing 2 times successively, each 10mL, with ethyl acetate layer is dry concentrate after, use silica gel column chromatography, ethyl acetate and sherwood oil (the two volume ratio is 1: 1) wash-out gets white solid two peptide fragment N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanines-L-proline methyl ester (Boc-N-Me-D-Phe-L-Pro-OMe) 1.47g, yield 96.7%.
Steps d-e: remove the segmental tertbutyloxycarbonyl protecting group of above-mentioned dipeptides, make products therefrom and N-(9-fluorenes methoxy carbonyl acyl group)-glycine condensation, get tripeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-glycine-N-methyl D-phenylalanine-L-proline methyl ester.During operation, above-mentioned 150mg (0.38mmol) Boc-N-Me-D-Phe-L-Pro-OMe is dissolved in the system of HCl/EtOAc that 3mL concentration is 4M, reaction is 30 minutes under the room temperature.After TLC shows that reaction finishes; underpressure distillation removes and desolvates; adding ether in residue heavily steams 2 times; get the white solid powder; directly be dissolved in 4mL N after the vacuum-drying; in the mixing solutions of dinethylformamide and methylene dichloride (the two volume ratio is 1: 3); add 65.0mg (0.77mmol) sodium bicarbonate; after 10 minutes; add 137.0mg (0.46mmol) N-(9-fluorenes methoxy carbonyl acyl group)-glycine, 88.0mg (0.46mmol) EDC.HCl and 63.0mg (0.46mmol) HOAt successively, ice bath returned to room temperature reaction 12 hours after 30 minutes.The pressure reducing and steaming methylene dichloride; add 100mL acetic acid ethyl dissolution residue; successively with 10% aqueous citric acid solution wash, 5% sodium bicarbonate aqueous solution and saturated common salt washing 2 times; each 10mL; with ethyl acetate layer is dry concentrate after; use silica gel column chromatography; (the two volume ratio is 1: 4-1: 1) wash-out with ethyl acetate and sherwood oil; get white solid tripeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-glycine-N-methyl D-phenylalanine-L-proline methyl ester (Fmoc-Gly-N-Me-D-Phe-L-Pro-OMe) 217.0mg, yield 99.0%.
Step f-g: the gained tripeptide fragment is removed 9-fluorenes methoxy carbonyl acyl group protecting group, with N-(tertbutyloxycarbonyl)-L-Isoleucine condensation, obtain the tetrapeptide fragment again.During operation, 927mg (1.63mmol) Fmoc-Gly-N-Me-D-Phe-L-Pro-OMe is dissolved in the mixed solvent of 30mL acetonitrile and diethylamine (the two volume ratio is 1: 1), room temperature reaction 2 hours, concentrating under reduced pressure removes and desolvates, then residue is dissolved in reconcentration in the methylene dichloride, repetitive operation 2 times, get thickness oily matter, after the vacuum-drying, be dissolved in the 30mL methylene dichloride, add N-(tertbutyloxycarbonyl)-L-Isoleucine 471.0mg (1.96mmol), ice bath is cooled to 0 ℃, add sodium bicarbonate 164.6mg (1.96mmol) successively, HOAt 266.8mg (1.96mmol) and EDC.HCl375.8mg (1.96mmol), ice bath reaction 30 minutes returned to room temperature reaction 12 hours.Follow the pressure reducing and steaming methylene dichloride, add 200mL acetic acid ethyl dissolution residue, use 10% aqueous citric acid solution successively, 5% sodium bicarbonate aqueous solution and saturated common salt water washing 2 times, each 10mL, with ethyl acetate layer is dry concentrate after, use silica gel column chromatography, (the two volume ratio is 1: 2-2: 1) wash-out with ethyl acetate and sherwood oil, get white foam shape solid tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester (Boc-L-Ile-Gly-N-Me-D-Phe-L-Pro-OMe) 730.0mg, yield is 80.0%.The segmental structural characterization of tetrapeptide is as follows: the migration value is R
f=0.26 (petroleum-EtOAc, 1: 1); Specific rotatory power is [α]
21 D=+19.3 ° (c 0.4, CHCl
3); The proton nmr spectra data are
1H NMR (CDCl
3, 600MHz) δ: 7.18-7.29 (m, 5H, Ar
H), 6.81 (brt, 1H, N
H-Ile), 5.58 (t-like, 1H, J=7.7,7.4Hz, α-CH-Phe), 4.42 (dd, 1H, J=8.2,6.0Hz, α-CH-Pro), 4.05-4.13 (m, 2H, α-CH-Ile, CH
2-a-Gly), 3.91 (dd, 1H, J=17.4,3.7Hz, CH
2-b-Gly), 3.73 (s, 3H, COOCH
3), 3.31-3.42 (m, 2H, δ-CH
2-Pro), 3.26-3.30 (m, 1H, β-CH
2-a-Phe), 2.99 (s, 3H, N-CH
3), 2.84 (dd, 1H, J=13.7,7.3Hz, β-CH
2-b-Phe), 2.13-2.20 (m, 1H, β-CH
2-a-Pro), 1.81-1.97 (m, 4H, β-CH
2-b-Pro, β-CH-Ile, γ-CH
2-Pro), 1.44 (m, 10H, γ-CH
2-a-Ile, (CH
3)
3), 1.09-1.13 (m, 1H, γ-CH
2-b-Ile), 0.89-0.93 (m, 6H, γ-CH
3-Ile, δ-CH
3-Ile); The carbon-13 nmr spectra data are
13C NMR (CDCl
3) δ: 172.5,171.5,167.9,137.0,129.4,128.6,128.4,126.8,126.7,79.9,59.3,59.0,56.2,52.3,46.8,41.2,37.5,35.0,29.7,28.8,28.3,25.0,24.6,15.7,11.6; Mass-spectrometric data is ESIMS:calced for C
29H
44N
4O
7[M+H]
+561.3; Found 561.4.
The preparation of (2.N-9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine, it is respectively as follows step by step:
At first with N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-D-glutamine is a raw material; by Fredinger method De Dao the oxazolidone intermediate; this intermediate is reductive ring open under trifluoroacetic acid and triethyl silicane condition, and removes trityl-protecting group and obtain N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine.During operation, with 2.00g (3.28mmol) N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-D-glutamine joins in the 100mL there-necked flask that water trap is housed, and adds 3mLN, dinethylformamide, stirring and dissolving, add toluene 60mL, add a hydration tosic acid 38mg and Paraformaldehyde 96 3.2g then successively, temperature rising reflux reaction 6 hours.Then concentrating under reduced pressure steams and desolventizes, residue is dissolved in the 200mL ethyl acetate again, organic layer is extremely neutral with saturated sodium bicarbonate aqueous solution washing, ethyl acetate layer is dry concentrated, use silica gel column chromatography, ethyl acetate and sherwood oil (the two volume ratio is 1: 2) wash-out gets Bai look Gu Ti oxazolidone intermediate 1.16g, and yield is 57%.Then (0.02mmol) oxazolidone intermediate is dissolved in the 14mL chloroform, adds 1.2mL triethyl silicane and 14mL trifluoroacetic acid then successively with 1.16g.Reaction solution at room temperature stirred 2 days.Concentrating under reduced pressure removes and desolvates then; residue is dissolved in reconcentration in the methylene dichloride; repetitive operation 3 times; use silica gel column chromatography; mixing solutions wash-out with chloroform and methyl alcohol (the two volume ratio is 10: 1); get white solid N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine (Fmoc-N-Me-D-Gln) 410mg, yield 57%.
3.L-2-the hydroxy-3-methyl valeric acid-leucic preparation of O-benzyl ester-L-, it is respectively as follows step by step:
Step a: make the L-Isoleucine obtain L-2-hydroxy-3-methyl valeric acid by the method for azo intermediate hydrolysis.During operation, it is in the dilute sulfuric acid aqueous solution of 1M that 4.0g (31mmol) L-Isoleucine is joined the 10mL concentration that is cooled to-5 ℃ in the cryosel bath, slowly dropwise adds the 60mL aqueous solution that contains 3.6g (52mmol) Sodium Nitrite then under cryosel is bathed.Cryosel was bathed reaction down after 2 hours, room temperature reaction 16 hours.With the reaction solution extracted with diethyl ether, dry concentrate yellow oil, add 1mL acetone, under-78 ℃ of conditions, grind, sticky solid, vacuum-drying gets light yellow solid L-2-hydroxy-3-methyl valeric acid (Hmp) 0.77g, yield 19.3%.
Step b: in the presence of toluene, benzylalcohol and tosic acid, refluxing obtains the leucic tosilate of O-benzyl ester-L-with the L-leucine.During operation, in the 50mL reaction flask that water trap and reflux condensing tube are housed, add 2.62g (20mmol) L-leucine, a hydration tosic acid 4.56g (24mmol), add 20mL toluene and benzylalcohol 8.35g (77.2mmol) then, stop heating behind the back flow reaction 6h.After temperature of reaction system is gradually reduced to room temperature, add 35mL ether and 35mL sherwood oil, alcohol-ether system recrystallization is used in the crystallization that suction filtration is separated out again, suction filtration obtains the white continuous shape solid O-leucic tosilate of benzyl ester-L-(L-Leu-OBn.TsOH) 5.33g, yield 67.8%.
Step c: condensation under the catalysis of condensing agent obtains L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-L-leucine with above-mentioned leucic tosilate of O-benzyl ester-L-and L-2-hydroxy-3-methyl valeric acid.During operation, 394mg (1.00mmol) L-Leu-OBn.TsOH is dissolved in the 10mL anhydrous methylene chloride, add acid binding agent N-methylmorpholine 133 μ L (1.20mmol), behind the ice bath 10 minutes, add Hmp132mg (1.00mmol), condensing agent HOAt 164mg (1.20mmol) and EDC.HCl 230mg (1.20mmol) successively, behind the ice bath 30 minutes, room temperature reaction spends the night.The pressure reducing and steaming methylene dichloride, add 200mL acetic acid ethyl dissolution residue, respectively wash 2 times with 10% aqueous citric acid solution, 5% sodium bicarbonate aqueous solution and saturated aqueous common salt successively, each 10mL, with ethyl acetate layer is dry concentrate after, use silica gel column chromatography, with ethyl acetate and sherwood oil (the two volume ratio is 1: 2) wash-out, get oily matter 250mg L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-L-leucine (Hmp-L-Leu-OBn), yield 74%.
4. the preparation of biologically active peptides Tasiamide-D-Gln, concrete respectively as follows step by step:
Step a-b: remove the segmental tertbutyloxycarbonyl protecting group of above-mentioned tetrapeptide; with N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine condensation, obtain pentapeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester then.During operation, Boc-L-Ile-Gly-N-Me-D-Phe-L-Pro-OMe is dissolved in the 4mL methylene dichloride with 100mg (0.18mmol) tetrapeptide fragment, adds 4mL trifluoroacetic acid and stirring reaction under the room temperature 2 hours.Concentrating under reduced pressure removes and desolvates then, and residue is dissolved in reconcentration in the methylene dichloride, and repetitive operation 2 times gets thickness oily matter.After the vacuum-drying, be dissolved among the 10mL exsiccant THF, add Fmoc-N-Me-D-Gln 98.8mg (0.26mmol), ice bath is cooled to 0 ℃, add N-methylmorpholine 48 μ L (0.43mmol), HOAt 58.0mg (0.43mmol) and EDC.HCl 82.0mg (0.43mmol) more successively, ice bath reacted after 30 minutes, room temperature reaction 20 hours.Pressure reducing and steaming solvent then; add 100mL acetic acid ethyl dissolution residue; use 10% aqueous citric acid solution successively; 5% sodium bicarbonate aqueous solution and saturated aqueous common salt are respectively washed 2 times; each 5mL; with ethyl acetate layer is dry concentrate after; use silica gel column chromatography; with chloroform and methanol mixed solvent (the two volume ratio is 20: 1) wash-out; get white foam shape solid pentapeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester (Fmoc-N-Me-D-Gln-L-Ile-Gly-N-Me-D-Phe-L-Pro-OMe) 143.6mg, yield is 97.9%.The segmental structural characterization of pentapeptide is as follows: the migration value is R
f=0.61 (CHCl
3-MeOH, 5: 1); Specific rotatory power is [α]
21 D=+13.0 ° (c 0.2, CHCl
3); The proton nmr spectra data are
1H NMR (CDCl
3, 600MHz) δ: 7.77 (d, 2H, J=7.3Hz, 4,5-C
H-Fluorenyl), 7.60 (d, 2H, J=6.6Hz, 1,8-C
H-Fluorenyl), 7.40 (t-like, 2H, J=7.7,7.3Hz, 3,6-C
H-Fluorenyl), 7.32 (m, 2H, 2,7-C
H-Fluorenyl), 7.17-7.23 (m, 5H, Ar
H), 6.97 (brs, 1H, N
H-Ile), 6.83 (brs, 1H, NH-Gly), 5.92 (brs, 1H, NH
2-a-Gln), 5.61 (brs, 1H, NH
2-b-Gln), 5.50 (t-like, 1H, J=7.3,6.6Hz, α-CH-Phe), 4.67 (brs, 1H, α-CH-Gln), 4.45-4.52 (m, 2H, CH
2-Fluorenyl), 4.40 (dd, 1H, J=8.8,5.8Hz, α-CH-Pro), 4.32 (brs, 1H, α-CH-Ile), 4.28 (brt, 1H, 9-CH-Fluorenyl), 4.06 (dd, 1H, J=17.9,4.8Hz, CH
2-a-Gly), 3.83 (d, 1H, J=16.5Hz, CH
2-b-Gly), 3.71,3.73
*(s, 3H, COOCH
3), 3.31 (t, 2H, J=6.2Hz, δ-CH
2-Pro), 3.25 (dd, 1H, J=13.6,8.0Hz, β-CH
2-a-Phe), 2.95,2.96
*(s, 3H, N-CH
3-Gln), 2.84 (s, 3H, N-CH
3-Phe), 2.80 (dd, 1H, J=13.9,7.3Hz, β-CH
2-b-Phe), 2.30-2.33 (m, 1H, β-CH
2-a-Gln), 2.10-2.20 (m, 3H, γ-CH
2-Gln, β-CH
2-a-Pro), 2.04 (brs, 1H, β-CH
2-b-Gln), 1.76-1.93 (m, 4H, β-CH
2-b-Pro, β-CH-Ile, γ-CH
2-Pro), 1.41-1.43 (m, 1H, γ-CH
2-a-Ile), 1.11 (brs, 1H, γ-CH
2-b-Ile), 0.87 (t, 6H, J=7.3Hz, γ-CH
3-Ile, δ-CH
3-Ile) (* represents rotational isomer); The carbon-13 nmr spectra data are
13C NMR (CDCl
3, 150MHz) δ: 172.5,167.9,143.9,143.8,141.3,136.9,129.4,128.4,127.8,127.1,126.7,125.0,120.1,68.0,59.1,58.9,58.0,56.3,52.3,47.2,46.8,41.1,37.0,35.0,32.2,29.8,29.7,28.8,25.0,24.8,24.1,15.6,11.3; Mass-spectrometric data is ESIMS:calced forC
45H
56N
6O
9[M+H]
+824.5; Found 825.5.
Step c-e: make above-mentioned pentapeptide fragment remove 9-fluorenes methoxy carbonyl acyl group protecting group and obtain an intermediate; L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-L-leucine is removed benzyl obtain L-2-hydroxy-3-methyl valeric acid-L-leucine under the condition of palladium preparation hydrogenolysis, then obtain biologically active peptides Tasiamide-D-Gln of the present invention with the condensation under the condition of condensing agent of above-mentioned intermediate.During operation, 20.0mg (0.02mmol) pentapeptide fragment Fmoc-N-Me-D-Gln-L-Ile-Gly-N-Me-D-Phe-L-Pro-OMe is dissolved in the mixed solvent of 4mL acetonitrile and diethylamine (the two volume ratio is 1: 1), reaction is 1 hour under the room temperature, after TLC shows that reaction finishes, the evaporate to dryness reaction soln, vacuum-drying gets pentapeptide fragment intermediate.In addition L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-L-leucine (Hmp-L-Leu-OBn) 100.0mg (0.30mmol) is dissolved in (the two volume ratio is 1: 4) in ethyl acetate and the ethanol mixed solvent, the 10% palladium carbon that adds catalytic amount, filled the hydrogen balloon hydrogenolysis 16 hours, TLC shows that reaction finishes, help with diatomite and to filter palladium carbon, evaporate to dryness filtrate, add the 10mL methylene dichloride, evaporate to dryness gets white solid L-2-hydroxy-3-methyl valeric acid-L-leucine (Hmp-L-Leu), and vacuum-drying 1 hour.Above-mentioned two kinds of intermediates that remove protecting group all are dissolved in the 20mL anhydrous methylene chloride; behind the ice bath 10 minutes; add condensing agent EDC.HCl 69.6mg (0.36mmol) successively; HOAt 49.4mg (0.36mmol) and N-methylmorpholine 44.0mg (0.36mmol); the ice bath reaction returned to room temperature reaction and spends the night after 30 minutes.Pressure reducing and steaming methylene dichloride then, add 50mL chloroform dissolved residue, use 10% aqueous citric acid solution, 5% sodium bicarbonate aqueous solution and saturated common salt water washing 2 times then successively, each 5mL, with organic phase is dry concentrate after, use silica gel column chromatography, with chloroform and methyl alcohol (the two volume ratio is 20: 1) wash-out, get colorless solid Tasiamide-D-Gln 10.0mg, yield 49.7%.The structural characterization of Tasiamide-D-Gln is as follows: the migration value is R
f=0.18 (CHCl
3-MeOH, 20: 1); Specific rotatory power is [α]
21 D=+15 ° (c 0.4, CHCl
3), the proton nmr spectra data are
1H NMR (CDCl
3, 600MHz) δ: 7.18-7.27 (m, 5H, Ar
H), 7.16 (d, 1H, J=8.7Hz, N
H-Leu), 7.09 (d, 1H, J=9.2Hz, N
H-Ile), 7.02 (t, 1H, J=4.1Hz, N
H-Gly), 5.98 (brs, 1H, NH
2-a-Gln), 5.63 (brs, 1H, NH
2-b-Gln), 5.53 (t-like, 1H, J=8.2,6.9Hz, α-CH-Phe), 5.05 (t-like, 1H, J=7.8,7.3Hz, α-CH-Gln), 4.94 (dt, 1H, J=10.6,6.4Hz, α-CH-Leu), 4.73 (d, 1H, J=4.1Hz, β-O
H-Hmp), 4.38 (dd, 1H, J=8.7,5.5Hz, α-CH-Pro), 4.30 (dd, 1H, J=9.1,6.8Hz, α-CH-Ile), 4.08 (dd, 1H, J=17.4,4.6Hz, CH
2-a-Gly), 3.83 (dd, 1H, J=17.4,3.7Hz, CH
2-b-Gly), 3.73
*, 3.71,3.66
*(s, 3H ,-COOC
H 3), 3.36 (m, 1H, δ-CH
2-a-Pro), 3.29 (m, 1H, δ-CH
2-b-Pro), 3.29 (m, 1H, β-CH
2-a-Phe), 3.01 (s, 3H, N-C
H 3-Gln), 2.99 (s, 3H, N-C
H 3-Phe), 2.82 (m, 1H, β-CH
2-b-Phe), 2.31 (m, 1H, β-CH
2-a-Gln), 2.23 (m, 1H, β-CH
2-a-Leu), 2.19-2.23 (m, 2H, γ-CH
2-Gln), 2.12 (m, 1H, β-CH
2-a-Pro), 2.00 (m, 1H, β-CH
2-b-Gln), 1.93 (m, 1H, γ-CH
2-a-Pro), 1.77-1.87 (m, 3H, β-CH
2-b-Pro, β-CH-Ile, γ-CH
2-b-Pro, β-CH-Hmp), 1.55-1.66 (m, 2H, γ-CH-Leu, β-CH
2-b-Leu), 1.40-1.47 (m, 2H, γ-CH
2-a-Hmp, γ-CH
2-a-Ile), 1.23 (m, 1H, γ-CH
2-b-Hmp), 1.11 (m, 1H, γ-CH
2-b-Ile), 0.96 (t-like, 9H, J=5.9,5.0Hz, γ-CH
3-Hmp, δ-CH
3-Leu
1, δ-CH
3-Leu
2), 0.85-0.90 (m, 9H, δ-CH
3-Hmp, γ-CH
3-Ile, δ-CH
3-Ile) (* represents rotational isomer); The carbon-13 nmr spectra data are
13C NMR (CDCl
3, 150MHz) δ: 174.5 (C-31), 174.2 (C-29), 174.1 (C-37), 172.5 (C-1), 171.4 (C-19), 169.7 (C-25), 167.9 (C-7), 167.7 (C-17), 136.9 (C-10), 129.4 (C-11,15), (128.4 C-12,14), 126.7 (C-13), 76.5 (C-38), 58.9 (C-2), 57.8 (C-20), 56.4 (C-26), 56.2 (C-8), 52.3 (C-6), 47.2 (C-32), 46.8 (C-5), 41.1 (C-18), 41.0 (C-33), 38.3 (C-39), 37.0 (C-21), 35.1 (C-9), 32.2 (C-28), 31.1 (C-30), 29.7 (C-16), 28.8 (C-3), 24.9 (C-4,34), 24.7 (C-22), 23.8 (C-40), 23.1 (C-27), 23.0 (C-35), 22.0 (C-36), 15.6 (C-23), 15.5 (C-41), 11.8 (C-42), 11.2 (C-24); The high resolution mass spectrum data are HRMS (ESI) calcd for C
42H
68N
7O
10[M+H]
+830.5028 found 830.5045.
Palladium preparation under the condition of the hydrogenolysis described in the present invention is palladium-carbon or palladium hydroxide.Described condensing agent is hydrochloride, 1-hydroxyl-7-azo benzotriazole or the 1-hydroxy benzo triazole of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide.Described N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-glutamine is N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-L-glutaminate or N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-D-glutamine.Described leucine is L-leucine or D-leucine.Mol ratio when described L-2-hydroxy-3-methyl valeric acid-L-leucine and pentapeptide fragment intermediate carry out condensation is 1: 1.2～1: 5.0.
The structural formula of biologically active peptides Tasiamide-D-Gln of the present invention is:
Determine that for further the three-dimensional absolute configuration of the synthetic biologically active peptides Tasiamide-D-Gln of institute of the present invention is (1S, 8R, 20S; 26R, 32S, 38S); the inventor has also synthesized three-dimensional absolute configuration and has been (1S, 8R, 20S; 26S; 32S, Tasiamide 38S), both preparation methods are identical; only in the process of synthetic N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine, the former adopts N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-D-glutamine is a raw material, and Tasiamide adopts N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-L-glutaminate is a raw material.The structural characterization of synthetic Tasiamide of the present invention: the migration value is R
f=0.18 (CHCl
3: MeOH=20: 1); Specific rotatory power is [a]
D 21=-12.6 ° (c 0.4, CHCl
3); The proton nmr spectra data are
1H NMR (CDCl
3): δ 7.19-7.35 (m, 5H, ArH), 6.99 (brs, 1H, NH-Gly), 6.80,6.29 (2 brs, 2H, NH
2-Gln), 5.52 (t-like, 1H, J=7.3,7.8Hz, α-CH-Phe), 5.11 (t, 1H, J=7.1Hz, α-CH-Gln), 4.94 (m, 1H, OH-Hmp), 4.81 (brs, 1H, α-CH-Leu), 4.41 (dd, 1H, J=8.3,5.5Hz, α-CH-Pro), 4.28 (t, 1H, J=7.3Hz, α-CH-Ile), 4.13,3.89 (2dd, 2H, J=17.4,4.6Hz, α-CH
2-Gly), 3.72 (s, 3H, COOCH
3), 3.34 (m, 2H, δ-CH
2-Pro), 3.28,2.81 (2dd, 2H, J=13.7,8.3Hz, β-CH
2-Phe), 3.13 (s, 3H, N-CH
3-Gln), 3.00 (s, 3H, N-CH
3-Phe), 1.80-2.99 (m, 10H, γ-CH
2-Gln, β-CH
2-Gln, β-CH
2-Pro, γ-CH
2-Pro, β-CH-Ile, β-CH-Hmp), 1.10-1.75 (m, 7H, γ-CH-Leu, β-CH
2-Leu, γ-CH
2-Ile, γ-CH
2-Hmp), 0.98 (d, 3H, J=6.9Hz, γ-CH
3-Hmp), 0.96,0.95 (2d, 6H, J=7.0Hz, 2 δ-CH
3-Leu), 0.89 (t, 3H, J=7.3Hz, δ-CH
3-Hmp), 0.87 (d, 3H, J=6.9Hz, γ-CH
3-Ile), 0.85 (t, 3H, J=7.3Hz, δ-CH
3-Ile); The carbon-13 nmr spectra data are
13C NMR (CDCl
3) δ: 175.5 (C-37), 174.1 (C-29), 174.0 (C-31), 172.5 (C-1), 171.2 (C-19), 170.4 (C-25), 167.9 (C-7), 167.9 (C-17), 137.0 (C-10), 129.4 (C-11,15), 128.5 (C-12,14), 126.8 (C-13), 76.2 (C-38), 59.0 (C-2), 58.0 (C-20), 56.5 (C-8), 55.4 (C-26), 52.3 (C-6), 47.8 (C-32), 46.8 (C-5), 41.2 (C-18), 41.0 (C-33), 38.6 (C-39), 36.5 (C-21), 35.0 (C-9), 31.5 (C-28), 31.0 (C-30), 29.9 (C-16), 28.8 (C-3), 25.0 (C-22), 24.9 (C-34), 24.4 (C-4), 23.9 (C-27), 23.4 (C-40), 23.2 (C-35), 21.4 (C-36), 15.7 (C-23), 15.5 (C-41), 11.8 (C-42), 11.3 (C-24); The high resolution mass spectrum data are HRMS (ESI): calcd for C
42H
68N
7O
10[M+H]
+830.5028 found 830.5035.
The carbon spectrum of synthetic biologically active peptides Tasiamide of the present invention and Tasiamide-D-Gln spectral data and natural product is compared, and as shown in drawings, wherein Fig. 1 is synthetic of the present invention (1S, 8R, 20S, 26S, 32S, the 38S) difference of Tasiamide and natural product carbon spectrum chemical shift, Fig. 2 is synthetic of the present invention (1S, 8R, 20S, 26R, 32S, 38S) the difference of Tasiamide-D-Gln and natural product carbon spectrum chemical shift, standard value is the chemical displacement value of natural product.Differ bigger by accompanying drawing 1 synthetic Tasiamide of the present invention as can be seen and natural product carbon spectrum chemical displacement value, by accompanying drawing 2 Tasiamide-D-Gln and natural product carbon spectrum chemical displacement value very nearly the same as can be seen, the true stereo absolute configuration that this shows natural product should be (1S, 8R, 20S, 26R, 32S, 38S), be biologically active peptides Tasiamide-D-Gln of the present invention.
For further research Tasiamide-D-Gln steric configuration is to active influence, the inventor has also synthesized three-dimensional absolute configuration and has been (1S, 8R, 20S, 26S, 32R, Tasiamide-D-Leu 38S) and three-dimensional absolute configuration are (1S, 8R, 20S, 26R, 32R, Tasiamide-D-Gln-D-Leu 38S), preparation method and Tasiamide-D-Gln are similar, difference is when synthesizing Tasiamide-D-Gln (seeing specific embodiment), the N of use
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-glutamine is N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine, uses the L-leucine to reflux in the presence of toluene, benzylalcohol and tosic acid and obtains the leucic tosilate of O-benzyl ester-L-; And when synthesizing Tasiamide-D-Leu; then adopting N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-L-glutaminate and D-leucine respectively is raw material; when synthesizing Tasiamide-D-Gln-D-Leu, adopting N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl D-glutamine and D-leucine respectively is raw material.Both structural formulas are as follows:
The structural characterization of synthetic Tasiamide-D-Leu and Tasiamide-D-Gln-D-Leu is as follows: the migration value of Tasiamide-D-Leu is R
f=0.18 (CHCl
3: MeOH=20: 1); Specific rotatory power is [α]
22 D=-15.6 ° (c 0.4, CHCl
3); The proton nmr spectra data are
1H NMR (CDCl
3, 600MHz) δ: 7.37 (dd, 2H, J=12.5,8.1Hz, NH-Leu, NH-Ile), 7.15-7.28 (m, 5H, ArH), 7.08
*(t, 2H, J=7.9Hz, NH-Leu, NH-Ile), 7.00 (dd, 1H, J=8.4,4.4Hz, NH-Gly), 6.74 (brs, 1H, NH
2-a-Gln), 6.67
*(brs, 1H, NH
2-a-Gln), 6.44 (brs, 1H, NH
2-b-Gln), 5.74
*(brs, 1H, NH
2-b-Gln), 5.51,5.47
*(t, 1H, J=7.5Hz, α-CH-Phe), 5.12 (dd, 1H, J=9.7,5.7Hz, α-CH-Gln), 4.93
*(dd, 1H, J=9.0,6.0Hz, α-CH-Gln), 4.89-4.93 (m, 1H, α-CH-Leu), 4.75-4.79
*(m, 1H, α-CH-Leu), 4.40,4.39
*(dd, 1H, J=5.7,1.8Hz, α-CH-Pro), 4.28 (dd, 1H, J=8.0,6.6Hz, α-CH-Ile), 4.09-4.15 (m, 2H, β-OH-Hmp, CH
2-a-Gly), 3.97,3.95
*(d, 1H, J=3.7Hz, α-CH-Hmp), 3.90,3.87
*(dd, 1H, J=14.3,3.8Hz, CH
2-b-Gly), 3.72
*, 3.71,3.66
*, 3.65
*(s, 3H, COOCH
3), 3.30-3.36 (m, 1H, δ-CH
2-a-Pro), 3.23-3.29 (m, 2H, β-CH
2-a-Phe, δ-CH
2-b-Pro), 3.11
*, 3.08 (s, 3H, N-CH
3-Gln), 3.00,2.99
*(s, 3H, N-CH
3-Phe), 2.82,2.79
*(dd, 1H, J=6.8,2.0Hz, β-CH
2-b-Phe), 2.18-2.39 (m, 3H, β-CH
2-a-Gln, γ-CH
2-Gln), 2.11-2.17 (m, 1H, β-CH
2-a-Pro), 1.99-2.03 (m, 2H, β-CH
2-b-Gln, β-CH-Ile), 1.90-1.97 (m, 3H, γ-CH
2-a-Pro, β-CH-Ile
*, β-CH
2-b-Gln
*), 1.84-1.89 (m, 1H, β-CH
2-b-Pro), 1.78-1.83 (m, 2H, β-CH-Hmp, γ-CH
2-b-Pro), 1.69-1.74,1.60-1.62
*(m, 1H, γ-CH-Leu), 1.63-1.68,1.56-1.59
*(m, 1H, β-CH
2-a-Leu), 1.35-1.49 (m, 3H, γ-CH
2-a-Hmp, γ-CH
2-a-Ile, β-CH
2-b-Leu), 1.14-1.22 (m, 1H, γ-CH
2-b-Hmp), 1.04-1.12 (m, 1H, γ-CH
2-b-Ile), 0.97 (d, 3H, J=6.6Hz, γ-CH
3-Hmp), 0.92-0.95 (m, 6H, δ
1, δ
2-CH
3-Leu), 0.81-0.90 (m, 9H, δ-CH
3-Hmp, γ-CH
3-Ile, δ-CH
3-Ile); (* represents rotational isomer) carbon-13 nmr spectra data are
13CNMR (CDCl
3, 150MHz) δ: 174.7,174.6,174.2,174.1,174.0,173.6,172.5,171.1,171.0,170.3,168.3,168.2,167.9,167.8,136.9,129.4,129.3,128.4,126.8,126.7,76.3,75.9,59.0,58.2,57.9,56.5,56.4,54.8,52.3,48.0,46.8,41.5,41.1,39.1,38.4,36.5,36.0,35.0,34.9,32.0,31.9,31.2,30.5,29.9,29.8,29.7,28.8,25.0,24.8,24.6,23.9,23.6,23.3,21.6,21.2,15.7,15.6,15.5,15.4,11.8,11.3; The high resolution mass spectrum data are HRMS (ESI) calcd for C
42H
68N
7O
10[M+H]
+830.5028 found 830.5008.
The migration value of Tasiamide-D-Gln-D-Leu is R
f=0.18 (CHCl
3: MeOH=20: 1); Specific rotatory power is [α]
21 D=+18.5 ° (c 0.4, CHCl
3); The proton nmr spectra data are
1H NMR (CDCl
3, 600MHz) δ: 7.21-7.25 (m, 5H, Ar
H), 7.19 (m, 1H, N
H-Leu), 7.11 (brs, 1H, N
H-Ile), 7.02 (brs, 1H, N
H-Gly), 6.06 (brs, 1H, NH
2-a-Gln), 5.76 (brs, 1H, NH
2-b-Gln), 5.52 (dd, 1H, J=8.4,7.0Hz, α-CH-Phe), 5.05 (t, 1H, J=7.3Hz, α-CH-Gln), 4.93 (m, 1H, α-CH-Leu), 4.77 (brs, 1H, β-O
H-Hmp), 4.38 (dd, 1H, J=8.8,5.5Hz, α-CH-Pro), 4.30 (dd, 1H, J=8.8,6.6Hz, α-CH-Ile), 4.08 (dd, 1H, J=17.6,5.2Hz, CH
2-a-Gly), 3.82 (dd, 1H, J=17.2,3.7Hz, CH
2-b-Gly), 3.73
*, 3.71,3.66
*(s, 3H ,-COOC
H 3), 3.35 (m, 1H, δ-CH
2-a-Pro), 3.29 (m, 1H, δ-CH
2-b-Pro), 3.29 (m, 1H, β-CH
2-a-Phe), 3.04
*, 3.00 (s, 3H, N-C
H 3-Gln), 2.99 (s, 3H, N-C
H 3-Phe), 2.81 (m, 1H, β-CH
2-b-Phe), 2.30 (m, 1H, β-CH
2-a-Gln), 2.23 (m, 1H, β-CH
2-a-Leu), 2.17-2.21 (m, 2H, γ-CH
2-Gln), 2.12 (m, 1H, β-CH
2-a-Pro), 1.99 (m, 1H, β-CH
2-b-Gln), 1.92 (m, 1H, γ-CH
2-a-Pro), 1.76-1.87 (m, 4H, β-CH
2-b-Pro, β-CH-Ile, γ-CH
2-b-Pro, β-CH-Hmp), 1.51-1.66 (m, 2H, γ-CH-Leu, β-CH
2-b-Leu), 1.40-1.45 (m, 2H, γ-CH
2-a-Hmp, γ-CH
2-a-Ile), 1.22 (m, 1H, γ-CH
2-b-Hmp), 1.10 (m, 1H, γ-CH
2-b-Ile), 0.93-0.97 (m, 9H, γ-CH
3-Hmp, δ-CH
3-Leu
1, δ-CH
3-Leu
2), 0.84-0.90 (m, 9H, δ-CH
3-Hmp, γ-CH
3-Ile, δ-CH
3-Ile) (* represents rotational isomer); The carbon-13 nmr spectra data are
13C NMR (CDCl
3, 150MHz) δ: 174.5 (C-29), 174.3 (C-31), 174.1 (C-37), 172.5 (C-1), 171.5 (C-19), 169.8 (C-25), 167.9 (C-7), 167.7 (C-17), 136.9 (C-10), 129.4 (C-11,15), (128.4 C-12,14), 126.7 (C-13), 76.4 (C-38), 58.9 (C-2), 57.8 (C-20), 56.4 (C-26), 56.2 (C-8), 52.2 (C-6), 47.3 (C-32), 46.7 (C-5), 41.1 (C-18), 40.9 (C-33), 38.3 (C-39), 37.0 (C-21), 35.0 (C-9), 32.2 (C-28), 31.1 (C-30), 29.7 (C-16), 28.8 (C-3), 24.9 (C-4,34), 24.7 (C-22), 23.7 (C-40), 23.2 (C-27), 23.0 (C-35), 22.0 (C-36), 15.6 (C-23), 15.5 (C-41), 11.8 (C-42), 11.2 (C-24); The high resolution mass spectrum data are HRMS (ESI) calcd for C
42H
68N
7O
10[M+H]
+830.5028 found 830.5035.
The application of biologically active peptides of the present invention in suppressing growth of tumour cell.
Adopt sulphonyl rhodamine B (SRB) protein staining method, used tumor cell line comprises BEL-7402 people's liver cancer, A-549 people's lung cancer, LOVO people's intestinal cancer, KB human oral squama cancer, HL-60 human leukemia, P388 mouse leukemia, is 72h action time.Biologically active peptides Tasiamide-D-Gln of the present invention is as shown in the table to the inhibiting rate of above-mentioned each tumor cell line, provides the concentration of biologically active peptides of the present invention and the relation between each tumor cell line growth inhibition ratio in the table.
Biologically active peptides Tasiamide-D-Gln is to the growth inhibition ratio of different tumour cells
Claims (7)
1, a kind of biologically active peptides, the molecular formula that it is characterized in that it is C
42H
67N
7O
10, structural formula is:
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26R, 32S, in the time of 38S), stereostructural formula is
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26S, 32R, in the time of 38S), stereostructural formula is
In the formula 1,8,20,26,32 and 38 s' three-dimensional absolute configuration be (1S, 8R, 20S, 26R, 32R, in the time of 38S), stereostructural formula is
2, the preparation method of the described biologically active peptides of claim 1, it is characterized in that at first making the L-proline(Pro) in the presence of thionyl chloride and methyl alcohol, to methylate, obtain the hydrochloride of L-proline methyl ester, making N-tertbutyloxycarbonyl-D-phenylalanine carry out nitrogen under the effect of sodium hydride and methyl iodide methylates, obtain N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine, hydrochloride and the condensation under the effect of the hydrochloride of condensing agent 1-hydroxyl-7-azo benzotriazole and 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide of N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine with above-mentioned L-proline methyl ester, obtain two peptide fragment N-(tertbutyloxycarbonyl)-N-methyl D-phenylalanine-L-proline methyl esters, remove the segmental tertbutyloxycarbonyl protecting group of this dipeptides, again with N-(9-fluorenes methoxy carbonyl acyl group)-glycine condensation, obtain tripeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-glycine-N-methyl D-phenylalanine-L-proline methyl ester, remove the 9-fluorenes methoxy carbonyl acyl group protecting group of this tripeptide fragment, with N-(tertbutyloxycarbonyl)-L-Isoleucine condensation, obtain tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester again; Then make N
α-(9-+ fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-glutamine Sheng under the effect of Paraformaldehyde 96 and tosic acid is Chenged the oxazolidone intermediate, this intermediate reductive ring open and remove trityl-protecting group and obtain N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine in the presence of trifluoroacetic acid and triethyl silicane; Make the L-Isoleucine obtain L-2-hydroxy-3-methyl valeric acid then by the method for azo intermediate hydrolysis, leucine is refluxed in the presence of toluene, benzylalcohol and tosic acid obtain O-benzyl ester-leucic tosilate, the condensation under the effect of the hydrochloride of condensing agent 1-hydroxyl-7-azo benzotriazole and 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide with above-mentioned L-2-hydroxy-3-methyl valeric acid and O-benzyl ester-leucine tosilate obtains L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-leucine; The last tertbutyloxycarbonyl protecting group that under the effect of trifluoroacetic acid, removes above-mentioned tetrapeptide fragment N-(tertbutyloxycarbonyl)-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; and with N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine condensation; obtain pentapeptide fragment N-(9-fluorenes methoxy carbonyl acyl group)-N-methyl-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; this pentapeptide fragment removes 9-fluorenes methoxy carbonyl acyl group protecting group; obtain intermediate N methyl-glutamine-L-Isoleucine-glycine-N-methyl D-phenylalanine-L-proline methyl ester; above-mentioned L-2-hydroxy-3-methyl valeric acid-O-benzyl ester-leucine after removing benzyl under the hydrogenolysis condition of palladium preparation, is obtained biologically active peptides of the present invention with above-mentioned intermediate condensation again.
3, the application of the described biologically active peptides of claim 1 in suppressing the kinds of tumor cells growth.
4, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that described palladium preparation is palladium-carbon or palladium hydroxide.
5, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that described N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-glutamine is N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-L-glutaminate or N
α-(9-fluorenes methoxy carbonyl acyl group)-N
γ-(trityl)-D-glutamine.
6, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that described leucine is L-leucine or D-leucine.
7, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that the mol ratio when described L-2-hydroxy-3-methyl valeric acid-L-leucine and intermediate carry out condensation is 1: 1.2～5.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617560A (en) * | 2011-01-30 | 2012-08-01 | 南京理工大学 | Preparation method of furilazole |
| CN103068794A (en) * | 2010-07-14 | 2013-04-24 | 韩国海洋研究院 | Method of synthesizing ramalin |
| CN112174854A (en) * | 2020-10-16 | 2021-01-05 | 上海吉奉生物科技有限公司 | Process for preparing (S) -2- ((9H-fluorene-9-methoxy carbonyl) methylamino) -5-amino-5-oxo pentanoic acid |
-
2008
- 2008-05-17 CN CNA2008100162496A patent/CN101274958A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103068794A (en) * | 2010-07-14 | 2013-04-24 | 韩国海洋研究院 | Method of synthesizing ramalin |
| CN103068794B (en) * | 2010-07-14 | 2015-07-22 | 韩国海洋研究院 | Method of synthesizing ramalin |
| CN102617560A (en) * | 2011-01-30 | 2012-08-01 | 南京理工大学 | Preparation method of furilazole |
| CN112174854A (en) * | 2020-10-16 | 2021-01-05 | 上海吉奉生物科技有限公司 | Process for preparing (S) -2- ((9H-fluorene-9-methoxy carbonyl) methylamino) -5-amino-5-oxo pentanoic acid |
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