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    1527252 Peptide cyclization process ARMOUR PHARMACEUTICAL CO 10 Sept 1975 [12 Sept 1974] 37226/75 Heading C3H Per se a non-cyclic peptide having an amino acid chain which contains two cysteine moieties, one of which has its sulphydryl function protected by an n-alkylthio group, whilst the second sulphydryl function may be protected by BZ may be prepared by a process which comprises coupling one cysteine moiety into an amino acid chain wherein the sulphydryl group of said moiety is protected with BZ, and coupling a second cysteine moiety into said chain, whilst protecting said sulphydryl function of said second moiety with an n-alkylthio group, and treating the peptide so formed with an acid to cleave said BZ without effecting said nalkylthio group. The non-cyclic peptide is held in solution (preferably aqueous or aqueous alcoholic) substantially free of oxygen (preferably in a stream of inert gas such as nitrogen), preferably at a pH of 5 to 10, until cyclization via a cystine linkage occurs. In the examples, condensation is effected using a solid phase synthesis or a polystyrene resin, with removal of the BOC group after each condensation; Ex. (A) (9) Boc-Gly-OH; (8) Boc-Leu-OH; (7) Boc-Pro-OH; (6) Boc- Cys(S-SEt)-OH; (5) Boc-Asn-OpNp; (4) Boc- Gln-OpNp; (3) Boc-Ile-OH; (2) Boc-Tyr(Bzl). OH; (1) Boc-Cys(S-pMeBzl)-OH to give Boc- Cys(S-pMeOBzl) - Tyr(Bzl) - Ile - Gln - Asn- Cys(S-SEt) - Pro - Leu - Gly - NHCH(C 6 H 5 )-X which is treated with HF to give H-Cys-Tyr- Ile - Gln - Asn - Cys(S-SEt) - Pro - Leu - Gly- NH 2 which, on cyclization, yields OXYTOCIN. Ex. (B) (32) Boc-Pro-OH; (31) Boc-Ala-OH; (30) Boc-Gly-OH; (29) Boc-Val-OH; (28) Boc-Gly-OH; (27) Boc-Ile-OH; (26) Boc-Ala- OH; (25) Boc-Thr(Bzl)-OH; (24) Boc-Gln- OpNp; (23) Boc-Pro-OH; (22) Boc-Phe-OH; (21) Boc-Thr(Bzl)-OH; (20) Boc-His(Z)-OH; (19) Boc-Phe-OH; (18) Box-Lys(Z)-OH; (17) Boo-Asn-OpNp; (16) Boc-Phe-OH; (15) Boc- Asp(OBzl)-OH; (14) Boc-Gln-OpNp; (13) Boc- Thr(Bzl)-OH; (12) Boc-Tyr(Bzl)-OH; (11) Boc- Thr(Bzl)-OH; (10) Boc-Gly-OH; (9) Boc-Leu- OH; (8) Boc-Met-OH; (7) Boc-Cys(S-SEt)-OH; (6) Boc-Thr(Bzl)-OH; (5) Boc-Ser(Bzl)-OH; (4) Boc-Leu-OH; (3) Boc-Asn-OpNp; (2) Boc- Gly-OH; (1) Boc-Cys(pMeOBzl)-OH which, on treatment as in Ex. (A) yields HUMAN CALCITONIN. Similarly there may be prepared: (1) H-Ala- Gly - Cys(SR) - Lys(V) - Asn - Phe - Phe - Trp- Lys(V) - Thr(Bzl) - Phe - Thr(Bzl) - Ser(Bzl)- Cys(3,4DiMeBzl) - OCH 2 X or H - Ala - Gly- Cys(SR) - Lys(V) - Asn - Phe - Phe - Trp- Lys(V) - Thr(Bz) - Phe - Thr(Bz) - Ser(Bz)- Cys(Bz) - OCH 2 X which yields SOMATOSTATIN. (2) H - Cys(Bzl) - Thr(Bz) - Phe - Gln - Asn- Cys(SR) - Pro - Arg(T) - Gly - NH - CH(C 6 H 5 ) -X which yields VASOPRESSIN. (3) H - Cys(Bz) Ser(Bz) - Asn - Leu- Ser(Bz) - Thr(Bz) - Cys(SR) - Val - Leu- Ser(Bz) - Ala - Tyr(W) - Trp - Arg(T) - Asn- Leu - Asn - Asn - Phe - His(V) - Arg(T) - Phe- Ser(Bz) - Gly - Met - Gly - Phe - Gly - Pro- Gly(Bz) - Thr(Bz) - Pro - NHCH(C 6 H 5 )-X which yields PORCINE CALCITONIN. (4) H - Cys(Bz) - Ser(Bz) - Asn - Leu- Ser(Bz) - Thr(Bz) - Cys(SR) - Val - Leu- Ser(Bz) - Ala - Tyr(W) - Trp - Lys(V) - Asp(Bz)- Leu - Asn - Asn - Tyr(W) - His(V) - Arg(T)- Phe - Ser(Bz) - Gly - Met - Gly - Phe - Gly- Pro - Glu(Bz) - Thy(Bz) - Pro - NHCH(C 6 H 5 ) -X which yields BOVINE CALCITONIN. (Bz, benzyl, 4-methoxybenzyl, 4-methylbenzyl, 3,4-dimethylbenzyl, 4-chlorobenzyl, 2,6 - dichlorobenzyl, 4-nitrobenzyl or benzhydryl; W, hydrogen Bz, benzyloxycarbonyl, 2 - chlorobenzyloxycarbonyl or 2 - bromobenzyloxycarbonyl; T, nitro or tosyl; V, benzyloxycarbonyl, 2 - chlorobenzyloxycarbonyl, 2 - bromobenzyloxycarbonyl or 3,4- dimethylbenzyloxycarbonyl; SR, n-alkylthio; X, polystyrene resin.)
    公开号:SU849998A3
    申请号:SU752171806
    申请日:1975-09-12
    公开日:1981-07-23
    发明作者:Лоренс Хьюз Джон；Кеннет Сейлер Джей；Чунг-Хуан Лиу Роберт
    申请人:Армор Фармасьютикал Компани (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to methods for producing cyclic peptides with a closed disulfide bridge, possessing biological activity that can be used in medicine. There is a known method of closing a disulfide ring in a peptide containing two cysteine residues by oxidizing the corresponding non-cyclic peptide 1. The main disadvantage of this method is that it requires the use of oxidizing agents, which can lead to contamination of the peptide with its oxidative degradation products and a decrease in the yield of the target product. In addition, during the oxidation of disulfide, in addition to the desired cyclic monomer, parallel and antiparallel dimers, cyclic or linear higher polymers are formed. As a result, the yield of the main product is reduced to 35%. The purpose of the invention is to increase the yield and simplify the process of obtaining cyclic peptides. The goal is achieved in that according to the method for producing cyclic peptides by closing a disulfide bridge in a non-cyclic peptide containing at least two cysteine residues, the original non-cyclic peptide is used, one of the cysteine residues of which contains a free sulfhydryl function and the second is a sulfhydryl function protected by - alkylthio group, and hold it in an aqueous solution free from oxygen at a pH of from 5 to 10, in a stream of inert gas. Moreover, a water-alcohol solution is used, nitrogen is used as an inert gas, and the process is carried out at pH 7.5. Non-cyclic peptides containing two cysteine amino acid residues, which are intermediate, have the structure 5R BZ 1 I ( A) j ,, a after acid treatment carried out for the cleavage of the protective groups, the string SR (A), where A is the amino acid residue; X - zero or integer / Cys - cysteine residue /
    R is an n-alkylthio group;
    Br - benzyl group or her
    derivative.
    The peptide has one cysteine residue with free sulfhydride new function, the second cysteine residue is searched for by an n-alkylthio group. Such peptides are kept in a solution, preferably single or alcoholic, at a pH of from 5 to 0 until a spontaneous regrouping takes place until the corresponding cyclic disulfide peptide is displaced with n-alkyl mercaptan.
    Example 1. Synthesis of oxytocin. Activation of the resin. 5 g of benzhydrylamine resin (BHA) 5 with an amine titer of 0.43 meq / g are placed in a reactor and treated with the following solvents, using each time 20 ml portions and a filter after each treatment: methylene chloride - 2 minutes, 20 chloroform - two times 2, min, 10% Triethylamine in chloroform - two times for 5 minutes, chloroform - 2 minutes, methylene chloride - three times for 2 minutes.
     9. Combination. 25 Mix the BHA resin, 20 ml of methylene chloride and 0.75 g for 10 minutes.
    (0.0043 mol) BOC-glycine. Then, 4.3 ml of a solution of dicyclohexylcarbodiimide in methylene chloride (1 meq of dicyclohexylcarbodiimide per 1 ml of solution) is charged to the reactor. The mixture is stirred b, h. The BOC-glycine-BHA resin is filtered off and subjected to the next successive 2 minute rinsings, each time with a filter; methylene chloride - (20 ml X 2), methyl alcohol (20 ml X 2), methylene chloride - (20 ml x X 2).
    Acetylation. With a mixture of 1.6 ml of acetic anhydride, 2.4 ml of triethylamine and 20 ml of chloroform are added to a 40 gum resin and stirred for 30 minutes. The reaction mixture is filtered and the resin is subjected to barely-washes, continuing each for 2 minutes, chloroform (20 ml X 2), methyl alcohol - (20 ml X 2), methylene chloride .- (20 ml x 3). Ningydrin test is negative. ate
    . Removing protection. The BOC-protected resin is mixed with 5-min with a mixture of 12 ml of trifluoroacetic acid and 12 ml of methylene chloride. Filter and mix the resin with a second mixture of 12 ml of trifluoroacetic acid and 12 ml of methylene chloride for 30 minutes. The mixture is filtered, and the resin is washed with the following solvents, 20 ml each: methylene chloride -2 pa-40 for 2 minutes, methyl alcohol - 2 times for 2 minutes, chloroform - 2 times for 2 minutes, 10% triethanolamine in chloroform 2 times rio 5 and 10 min, methylene chloride 2 times 2 min. five
    For a BHA-L-glycine resin, a titer of amine or glycine, amounting to 0.384 meq amine or glycine per gram of resin.
    Cycle 8. Combine and, E. L-Glycine gum, 20 ml of methylene chloride and 2.95 g (0.0038 mol) of BOC-L-leucine are stirred in water for J.O min. Then, 3.8 MP of a solution of dicyclohexylcarbodiimide in methylene chloride (1 mLs of dicyclohexylcarbodiimide (DCC1) per 1 ml of solution or only 0.0038 mol of DCC1) is added to the reactor. The mixture is stirred for 2 hours, then the reaction mixture is removed from the reactor and the resin is subjected to the following successive 2-minute wash, each filter: methylene chloride (20 ml X 2), methyl alcohol - (20 ml X 2), methylene chloride - (20 ml x 2). Ningydrin test is negative.
    .The protection is removed, in this cycle. The procedure for removing the protective groups is repeated, as in cycle 9.
    Cycle 7. The combination and deprotection is carried out as in cycle 8, but instead of the leucine derivative, 0.82 g (O., 0038 mol) of BOC-L-proline is used.
    Cycle 6. Combination and deprotection are carried out under cycle conditions 8. Acetylation as in cycle 9. 1.07 g (0.0038 mol) BOC-5-ethylthio-1-cysteine is used for the combination.
    Cycle. 5. Combination. Peptide resin from cycle b was washed twice, taking 20 ml of dimethylformamide. The resin is then stirred for 24 hours with a solution of 2.02 g (0.0057 mol) of BOC-L-asparagine-p-nitrophenyl ether in 25 ml of dimethylformamide. The reaction mixture is filtered and the peptide resin is subjected to successive two-minute washes of 20 ml each with the following solvents: dimethylformamide, methylene chloride, methanol, methylene chloride. The washings are separated by filtration. Ninhydrin test is negative.
    Removing protection. The method of removing the protective groups, as in cycle 8.
    Cycle 4. The combination is carried out under the conditions described in cycle 5. Acetylation is the same as in cycle 9. Deprotection of the protective groups is the same as in cycle 8. The following amino acid derivative is used: 2.09 g (0.0057 mol) n- nitrophenyl ester of BOC-L-glutamine.
    Cycle 3. The combination was carried out under the conditions described in cycle 8. The combination was repeated using a mixture of 10 ml of dimethylformamide and 10 ml of methylene chloride, and the same amount of amino acid and dicyclohexylcarbodiimide. Acetylation is carried out as in cycle 9. Disruption of the protective groups, as in cycle 8 O, 88 g (0.0038 mol) of BOC-L-isoleucine are used for the coupling reaction.
    Cycle 2. Combination. The peptide resin from cycle 3 is washed with two successive portions of 20 ml of dimethylformamide. Then the peptide resin is stirred for 10 minutes with a mixture of 2.11 g (0.0057 mol) of BOC-o-benzyl-1-tyrosine and 20 ml of dimethylformamide. Then, 5.7 ml of d of cyclohexylcarbodiimide in methylene chloride (equivalent to 0.0057 mol of OCC1) are added, and the mixture is stirred for 16 hours and filtered. The peptide resin is subjected to 2-minute washing each time with 20 m each with the following solvents 2 times each: dimethylformamide, methylene chloride, methanol, and methyl chloride. The combination was repeated, using half of the indicated number of amino acid derivatives and dicyclohexylcarbodiimide in methylene chloride with stirring for 6 hours. Acetylation. Conducted as in example 9. The removal of protective groups. The procedure is carried out as in Example 9. Cycle 1. The combination is carried out as in cycle 8. The combination is repeated using half the indicated amount of the amino acid derivative and didiclohexylcarbodiimide in methyl chloride. The removal of the protective groups is carried out as in cycle 9. In the first combination reaction, 1.3 g (0.038 mol) of vo-S-methoxybenzyl-L-cysteine is used. At the end of cycle 1, the peptide resin is sequentially washed with two portions of 20 ml of n-hexane. The peptide substance is removed from the reactor and dried in an electrovacuum oven at 1 mm Hg. within 24 hours. The weight of the blocked peptide resin oxytocin g. Cleavage with fluorine hydrogen Ohm. 2g of dried peptide resin and 2 ml of anisole are placed in a Teflon peaKTOpV equipped with a magnetic stirrer with a Teflon coating. The reactor is cooled in a bath with dry ice and acetone. 15 ml of gaseous hydrogen fluoride are condensed into the reactor. The mixture was stirred at 0 ° C in an ice bath for 1 hour. Hydrogen fluoride was evaporated under reduced pressure. The residue is triturated with 4-25 ml portions of ethyl acetate. The peptide is extracted from the resin with 2x50 ml portions of glacial acetic acid. The extract is lyophilized, 486 mg of cleaved peptide are obtained. Cyclization, peptide to oxytocin. 200 mg of dry pentide is dissolved in 50 ml of an oxygen-free distilled bath water with the addition of 1 ml of glacial acetic acid. The pH of the solution was adjusted to 7.5 with concentrated ammonium hydroxide. The mixture is stirred in a sealed vessel in a stream of nitrogen for 24 hours. By this time, mercaptan is no longer contained in the outgoing nitrogen. The content of ethyl mercaptan in the outgoing nitrogen is determined by passing a fasa current through the Ellman reagent solution. The pH of the reaction mixture was adjusted to 3.5 by the addition of glacial acetic acid. Lyophilized and get a solid residue. Purification of raw oxytocin. The solid residue is dissolved in 0.5N. acetic acid and clean, pass through a column filled with Sephadex G 25 (thin), and elute with 0.5N. acetic acid. The oxytocin fraction from this column is collected and lyophilized, 54 ml of a white precipitate are obtained. The precipitate was redissolved in 0.5N acetic acid and purified by gel filtration through Ce-fadex G-25 (thinJ, eluted with 0.5N acetic acid. The oxitocin fraction was collected, lyophilized, and a fluffy white residue was obtained. Product analysis showed the following amounts of 1mino acids: Gly 1.0; Leu 0.98; Pro 0.97; Asp 0.89; Glu 0.84; lie 0.74; Tyr 0.72. The theoretical amount should be 1.0. Cys is not determined, since this acid is destroyed in the analysis. The biological activity of the obtained product is 305.4 units per 1 mg. This method can also be applied Example 2. Synthesis of human calcite-onin. Activation of the resin. 4 g of benzhydrylamine resin (BHA) with amine titers of 0.55 meq / g is loaded into the reactor and treated with 20 ml of the following solvents, the filter after each washing. methylene chloride for 2 minutes , chloroform twice in 2 minutes, 10% triethyl-amine in chloroform twice in 5 minutes, chloroform - 2 minutes, methylene chloride three times in 2 minutes. Cycle 32. Combination. BHA resin, .20 ml of methylene chloride and 0.95 g (0.0044 mol) of BOC-L-proline. Mix 10 min. 4.4 ml of a methylene chloride solution of dicyclohexylcarbodiimide (1 meq OCC1 per 1 ml of solution) was added to the reactor and the mixture was stirred for 6 hours. The reaction mixture was filtered and the resin BOC-L-prolyl-BHA was subjected to successive washes for 20 minutes for 20 minutes solvent, each time the filter: methylene chloride - 2 times,., methyl alcohol - 2 times, methylene chloride - 2 times. Acetyl, ation. The resin is stirred with a mixture of 1.5 ml of triethylamine, 1 ml of acetic anhydride and 20 ml of chloroform for 2 hours. The reaction mixture is filtered, and the resin is washed successively for 2 minutes each with 20 ml of the following solvents. chloroform - 2 times. methyl alcohol - 2 times, and methylene chloride - 3 times. Ninhydrin test is negative. Removal of protective groups. The BOC-protected resin is mixed for 5 minutes with a mixture of 12.5 ml of fluorine-acetic acid and 12.5 ml of methylene chloride. The mixture is filtered and the resin is stirred with a second mixture consisting of 12.5 ml of trifluoroacetic acid and 12.5 ml of methylene chloride for 30 minutes. The reaction mixture is filtered and the resin is subjected to successive washing with 20 ml: methylene chloride 2 times for 2 minutes, methyl alcohol 2 times for 2 minutes, chloroform 2 times for 2 minutes, 10% triethylamine in chloroform 2 times for 10 minutes, chloroform 2 times 2 min, methylene chloride house 2 times 2 min. Resin L-Proline-VNL titrated for. adjusting the amine titer or proline. This value is 0.494 meq amine or proline per gram of resin, C and K l 31. Combination. L-prolyl resin, 20 ml of methylene chloride and 0.83 g (0.0044 mol) of VOSALANIN are mixed for 10 minutes. Then 4.4 ml of a solution of dicyclohexylcarbodiimide in methylene chloride (1 meq DCC1 per 1 ml of solution or only 0.0044 mol of DCC) was added to the reactor, and the mixture was stirred for 2 hours. The reaction mixture was filtered and the resulting BOC-1-alanyl-1 -prolyl-BHA, the resin is subjected to successive 3-minute washing with 20 ml each time to the filter: methylene chloride 2 times, methyl alcohol 2 times, methylene chloride 3 times. Ninhydrin samples are negative. Cycles 30-26. The combination and deprotection is carried out as in cycle 3.1, but the following amino acid derivatives are used instead of the alanine derivative: cycle 30-0.77 g (0.0044 mol) BOC-glycine, cycle 29 0.95 g (0.0044 mol ) BOC-L-valine, cycle 28 is the same substance as in cycle 30, cycle 27 is 1.02 g (0.0044 mole of BOC-L-isoleucine, cycle 26 is the same substance as in cycle .31. Cycle 25, Combination: The peptide resin from cycle 26 was washed twice with 20 ml portions of dimethyl form amide, then it was stirred for 10 minutes with a mixture of 2.04 g (0.0066 mol) of BOC-p-benzyl-1-threonine and 20 ml of dimethylformamide. 6.6 ml of dicyclohexylcarbodime are added. yes in methylene chloride (equivalent to 0.0066 mol of DCC1), and the mixture is stirred for 6 hours. The reaction mixture is filtered. The peptide resin is subjected to 2 min washes in 20 ml portions in the following solvents: dimethylformamide, methylene chloride, methanol, methylene sulfide Ninhydrinos and sample: Deprotection: As in cycle 32. Cycle 24: Negative treatment. The peptide resin from cycle 25 is washed twice with 20 ml portions of dimethylformamide. The resin is then stirred for 24 hours with a solution of 2.42 g (0.0066 mol) of BOC-L-glutamine p-nitrophenyl ester in 25 ml of dimethylformamide. The reaction mixture is filtered and the peptide resin is subjected to 2-minute washes with two successive 20 ml each. in the following solvents: dimethylformamide, methylene chloride, methanol, methylene chloride. Each solvent is filtered off. Ninhydrin test is negative. Removing protection. Conduct as in cycle 32. Cycle 23. Combination. The peptide resin from cycle 24 is mixed. 10 min with 1.42 g (0.0066 mol) of BOC-L-proline and 20 ml of methylene chloride. 6.6 ml of a solution of dicyclohexylcarbodiimide in methylene chloride (equivalent to 0.0066 mol of DCCI) are added and the mixture is stirred for 16 h. Reac. The mixture is filtered and the peptide resin is subjected to successive washes of 20 ml each, for 2 minutes with the following solvents: methylene chloride, methyl alcohol, methylene chloride. Each wash is filtered, the Ninhydrin test is negative. Removing protection. Conducted as in cycle 32. Cycle 22. Combination and deprotection are carried out as in cycle 23, but when combined, instead of BOC-L-proline, 1.75 g (0.0066 mol) of BOC-L-phenylalanine is used. Cycles 21 to 18. The combination and deprotection is as in cycle 31, but instead of the alanine derivative, the following amino acid derivatives are used: cycle 21 1, 36 g (0.0044 mol) BOC-o-benzyl-L-threonine cycle 20 - 1.71 g (0.0044 mol) of BOC-N- (jt) -carbobenzyloxy-1-histidine, cycle 19 1.17 g (0.0044 mol) of BOC-L-phenylalanine, cycle 18 - 1.67 g (0,0044 mol) of BOC-carbobenzyloxy-1-lysine. . . Cycle 17. The combination and deprotection is carried out as in cycle 24, but instead of the glutamine derivative, 2.33 g (0.0066 mol) of BOC-L-asparagine p-niftophenyl ester is used. Ts and ky s 16 and 15. The combination and removal of protection is carried out as in cycle 31, but instead of the alanyl derivative, the following amino acid derivatives are taken: cycle 16 - G, 17 g (0.0044 mol) OC-i-phenylalanine cycle 15 - 1.42 g (0.0044 mol) of OC-L-aspartic acid benzyl ester. Cycle 14. drive in the same way as cycle 24. Cycle 13. Carry out in the same way as cycle 21. Cycle 12. Combination and deprotection is carried out under the conditions described in cycle 15, but instead of the threonine derivative, they take 2.45 g (0.0066 mo BOC-o-benzyl-1-tyrosine, and stir the mixture for 16 hours. C and C 11. Carry out the same way as cycle 25. C and C s 10-7. Combination and removal of protection lead as in cycle 31, but the following amino acid derivatives are taken instead of BOC-L-alanine in the coupling reaction: cycle 10 - 0.77 g (0.0044 mol) BOC-glycine, cycle 9 1, 02 g (0.0044 mol) BOC-L-leucine cycle 8 - 1.1 g (0.0044 mol) of BOC-L-methionine, cycle 7 - 1.24 g (0.0044 mol) of BOC-5-ethyl io-1-cysteine. Cycle 6. They are the same as cycle 25. Cycles 5 and 4. The combination and deprotection is carried out as in cycle 31, but instead of BOC-L-alanine, the following amino acid derivatives are used cycle 5 - 1.3 g (0.0044 mol) of BOC-o-benzyl-1-serine, cycle 4 - 1.02 g (0.0044 mol) of BOC-L-leucine. Cycle 3. The conditions are the same as and in cycle 17. Cycles 2 and 1. The combination and deprotection is carried out as in cycle 31, but the following amino acid derivatives are used instead of BOC-L-alanine: cycle 2 - 0.77 g (0.0044 mol) BOC-glycine, cycle 1 - 1.5 g, JO, 0044 mole BOC-5-p-methoxybenzyl-1-cysteine. At the end of cycle 1, the pentide resin is washed twice with 20 ml portions of n-hexane. The peptide substance is discharged from the reactor and dried in an electrovacuum oven at 40 s and 0.11 mm Hg. within 24 hours. The weight of the blocked peptide resin of human calcitonin is 11 g. Splitting with fluoride hydrogen. 2g of dried peptide c) 1ola and 2 ml of aniso are placed in a heating reactor equipped with a teflon-coated magnetic stirrer and cooled in a bath with acetone and dry ice. 15 ml of gaseous hydrogen fluoride are condensed into the reactor. The mixture was stirred with an ice bath for 1 hour. Hydrogen fluoride was distilled off under reduced pressure. The residue is triturated with 4x25 m of ethyl acetate. The peptide is extracted from the resin with 2x50 ml of glacial acetic acid. The extracts are lyophilized and 1063 mg of cleaved peptide is obtained. Cyclization of peptide to human calcitonin. 1000 mg of crude peptide was dissolved in 250 ml of non-oxygenated distilled water with 1 ml of glacial acetic acid, the pH of the solution was adjusted to 7.5 with concentrated ammonium hydroxide. The mixture is stirred in a sealed vessel in a stream of nitrogen for 24 hours. By this time, ethyl mercaptan is no longer contained in the outgoing nitrogen. The content of ethyl mercaptan in the effluent nitrogen is determined by passing nitrogen through an Ellman reagent solution. The pH of the reaction mixture was adjusted to 3.2 by the addition of glacial acetic acid. After lyophilization, a solid product weighing 985 mg is obtained. Purification of raw human calcitonin. The solid was dissolved in 0.5N acetic acid and purified by gel chromatography on Sephadex G-25 (thin), eluted with 0.5N. with acetic acid, a fraction of human calcitonin from this column is collected and lyophilized; a white fluffy precipitate is obtained, which is dissolved in 0.05 M aqueous ammonium acetate (). The pH of the solution was adjusted to 5 and the solution was purified by ion exchange chromatography on an SR-Sephadex G-25 column. The product is eluted with ammonium acetate buffer. The fraction containing human calcitonin, twice lyophilized, get a fluffy white precipitate. It is established that this substance is biologically and chemically equivalent to the product described in the literature. Amino acid analysis (acid hydrolysis) gives the following amounts of amino acids (theoretical values are given in parentheses): Lys 1.02 (1); His 0.99 Asp 3.28 (3); Thz 5.21 (5); Gly 4.33 (4); 1.95 (2) 0.74 (1); Glu (1); Met Ala 2.03 (2); Val 1.04 0.86 (1); Me 1.07 (1); Leu 2.24 (2); tyr 0.7 (1); Phe 3.0 (3). The values for Pro and Cys are not determined. The biological activity is 100 MRC units per mg. This method can be applied in the synthesis of vasopressin, calcine calcitonin, bovine calcitonin. Example 3. Synthesis of salmon calcitonin. Activation of the resin. 5g of BHA resin with an amine titer of 0.61 meq / g are placed in the reactor and treated with solvents, each time taking 25 ml of the corresponding solvent and filter the reaction mass after each treatment: methylene chloride 2 minutes, chloroform - 2 minutes two times, 10 % triethylamine in chloroform 5 minutes two times, chloroform - 2 minutes, methylene chloride - g min three times. Cycle 32. Combination. BHA resin 25 ml, methylene chloride and 1.29 g (0.006 mol) of BOC-L-proline. stirred for 10 minutes, added 6.0 ml of a solution of dicyclohexylcarbodiimide (1 meq DCC1 per 1 ml of solution) in methylene chloride, and the mixture is stirred for 6 hours. BOC-Proline-BHA resin is removed and subjected to the following successive 2-minute washes: methylene chloride (2x25 ml ), methyl alcohol (2x25 ml) and again with methylene chloride (3x25 ml). After each wash, the resin is filtered. Acetylation. A mixture of 1.5 ml of triaethyl amine, 1 MP of acetic anhydride and 25 ml of chloroform is added to the resin and stirred for 2 hours, then filtered and the resin is washed with the following solvents: chloroform (2x25 ml), methyl alcohol (2x25 ml) , methylene chloride (3x25 ml). Each flush lasts 2 minutes. Removal of protecting groups. The BOC-protected resin is stirred for 5 minutes in a mixture of 15 ml of trifluoroacetic acid and 15 ml of methylene chloride, filtered, and again mixed with the same mixture of solvents for 30 minutes. Filtered and the resin washed with solvents, taking 25 ml of each: methylene chloride (2 x 25 ml), methyl alcohol (2 x 25 ml chloroform (2 x 25 ml). Each washing was continued for 2 minutes with 10% triethylamine in chloroform (2 x 25 ml) for 10 minutes chloroform and methylene chloride (2x25 ml). The last two washes are also continued for 2 minutes. For resin L-proline-BHA set the titer of amine or proline. This value is 0.55 meq amine or proline per gram of resin. Cycle 31. C oh et al. Mixture containing L-proline resin, 25 ml of methylene chloride and 1.7 g (0.0055 mol) of BOC-o-benzyl-1-threonine is mixed with 10 mi Then, 5.5 ml of a solution of dicyclohexylcarbodiimide (1 meq DCC1 per ml of solution or only 0.0055 mol of DCC1) in methylene chloride is added to the solution and the mixture is stirred for 2 hours. The reaction mixture is filtered and the resin is subjected the following consecutive 2-minute washes: methylene chloride (2x25 ml), methyl alcohol (2x25 ml), methylene chloride (3x25 ml). Ninhydrin reaction is negative. Unprotected g. p. n. Conducted under conditions described for cycle 32.. Cycles 30-27. The procedure of combining and removing protecting groups is used as in cycle 31, but instead of the third production they use the following amino acid derivatives of the cycle 30-0.97 g (0.0055 mol) of BOC-glycine; cycle 29 - 1.62 g (0.0055 mol) of BOC-o-benzyl-1-serine; cycle 28 is the same compound as in cycle 30; cycle 27 is the same compound as in cycle 31. Cycle 26. Combination, un. Peptide resin from cycle 27 washing with imethylformamide (3x25 ml). The resin is then stirred for 24 hours with a solution of 2.9 g (0.008 mol), BOC-L-asparagine p-nitrophenyl ester in 35 ml of dimethylformamide. The reaction mixture is filtered and the peptide resin is subjected to 2-minute washing with successively the following solvents: dimethylormamide, methylene chloride, methanol, ethyl chloride, each time taking 25 ml. The solvent is filtered off. Ninhydrin test is negative. Removal of protective groups. Carried out under the conditions described for cycle 32. Cycle 25. The combination and removal of the protecting groups is carried out under the conditions described for cycle 31, using the same substances in the same amounts. C and to l 24 .. Combination. The peptide resin from cycle 25 was washed, a1bt in two successive portions of 25 ml of dimethylformamide and stirred for 10 minutes with a mixture of 3.43 g (0.08 mol) of BOC-N-y-L-arginine in 25 ml of dimethylformamide. Then 8 ml of dicyclohexylcarbodiimide in methylene chloride (equivalent to 0.008 mol of DCC1) are added, the mixture is stirred for 6 hours and filtered. The peptide resin is subjected to two-minute washes with solvent solids mi-: dimethylformamide (2x25 ml), methylene chloride (2x25 ml), methyl alcohol (2x25 ml), methylene chloride (2x25 ml). Ninhydrin test is negative. Removal of protective groups. Carried out under the conditions described for cycle 32. Cycle 23. Combination. The peptide resin obtained in 24. is stirred for 10 minutes with 1.77 g (0.008 mol) of BOC-L-proline and 25 ml of methylene chloride. 8 ml of dicyclohexylcarbodiimide in methylene chloride (equivalent to 0.008 mol of DCCl is added; the mixture is stirred b, filtered and the peptide resin is subjected to two-minute washes with the following solvents: methylene chloride (2x25 ml), methyl alcohol (2x25 ml), methylene chloride (2x25 mL / mL). the resin is filtered. The ninhydrin reaction is negative. Removal of protecting groups. Carried out under the conditions described in cycle 32. Cycles 22 and 21. Conditions in which the combination and removal of protective groups are carried out are described in cycle 24, but in the combination reaction instead of B The following amino acid derivatives are used for C-L-proline: cycle 22 2, 97 g (0.008 mol) BOC-o-benzyl-L-tyrosine, cycle 21-2-247 g (Of008 mol) BOC-o-benzyl-1- threonine ..
    Cycle 20. The procedure is the same as that described in cycle 26, but instead of the aspartic derivative, 3.0 g (0.008 mol) of wo-L-glutamine p-nitrophenyl ester is used.
    Cycles 19-15, The procedure is the same as that described in cycle 31, but instead of threonine derivatives, the following amino acid derivatives are taken: Cl 19 - 1.37 g (0.0055 mol) BOC-L-leucine, cycle 18 -.2.09 g (0.0055 mol |) BOC-L-carbobenzyloxy-1-lysine / cycle 17 - 2.58 g (0.0055 mol BOC-N- (it) -carbobenzyloxy-1-histidine; cycle 16 - as in the cycle 19 / cycle 15 - 1.85 g (0.0055 mol) of BOC-L-glutamic acid y-benzyl ester.
    Cycle 14. Reaction conditions are described in cycle 20.
    Cycle 13. The procedure is the same as in Cycle 23, but instead of the prolon derivatives, 2.36 g (0.008 mol) of BOC-o-benzyl-L-serine are taken as a combination reaction.
    Cycles 12-9. The procedure is the same as in cycle 31, but the following amino acid derivatives are used instead of a threonine derivative in a combination reaction: cycle 12 is the same substance as in cycle 19; cycle 11 is the same substance as in cycle 13; cycle 10 is the same substance as in cycle 30} cycle 9 is the same substance, 41 o and in cycle 19.
    Cycle 8. Combination. The peptide resin obtained in cycle 9 was stirred for 10 minutes with 1.79 g (0.008 mol) of BOC-L-valine and 25 ml of methylene chloride. Then 8 ml of dicyclohexylcarbodiimide in methylene chloride (equivalent to 0.008 mol of DCC1) are added, and the mixture is stirred for 16 hours. The peptide resin is filtered and subjected to 2-minute washing with the following solvents; methylene chloride (2x25 ml), methyl alcohol (2x25 ml), methylene chloride (2x25 ml). After each wash, the resin is separated by filtration.
    Removal of protective groups. Conducted as described in cycle 32.
    Cycle 7. Method from cycle 31, but when combined instead of a threonine derivative, 1.55 g (0.0055 mol) of BOC-5-ethylthio-1-cysteine is used.
    Cycle 6. Substances and methods are described in cycle 31.
    Cycle 5. Substances and methods are described in cycle 29 ..
    Cycle 4. Substances and methods are described in cycle 19.
    Cycle 3. Substances and methods are described in cycle 26.;
    Cycle 2. Substances and methods are described in cycle 29.
    C and C L 1. The procedure is described in cycle 31, but instead of the threonine derivative is used. 1.88 g (0.0055 mol of BOC-5-p-methoxybenzyl-1-cysteine.
    At the end of cycle 1, the peptide resin is washed, n-hexane (2x25 ml) is filtered and dried in an electric vacuum oven at a pressure of 0.1 mm Hg. within 24 hours
    Dissipation. Hydrogen fluoride. 16 g of a mixture of dry peptide resin and 16 ml of anisole are placed in a Teflon reactor equipped with a Teflon-coated magnetic stirrer. : Reactor.- cooled in a bath of dry ice and acetone and condensed in it 100 ml of hydrogen fluoride. This mixture was stirred with an ice bath for 1 hour. Hydrogen fluoride was evaporated under reduced pressure. The residue is triturated in ethyl acetate (6x100 ml). The peptide is extracted with 800 ml of O, 1 M aqueous solution of acetic acid.
    Cyclization of peptide to salmon calcitonin. The aqueous acetic acid extract of the peptide obtained after cleaving the resin with hydrogen fluoride was diluted to 1.5 liters with 700 ml of distilled water, the pH of the solution was adjusted to 7.5 by the addition of concentrated ammonium hydroxide. The solution is stirred in a sealed vessel in a stream of nitrogen for 24 hours. During this time, all the split ethyl mercaptan is removed With a stream of nitrogen. The content of ethyl mecaptan in a stream of nitrogen is determined using Ellman's agent, the pH of the reaction mixture is adjusted to 5.0 with glacial acetic acid.
    Purification of raw salmon calcitonin. 1.5 l of the obtained solution with pH 5.0. Is concentrated using an SD-Sephadex 0-25 ion exchange column. 75 ml of concentrate are obtained in a 0.5 M solution of sodium chloride, it is desalted, cleaned by gel filtration through Sephadex G-25 (fine), eluted with 0.03 M aqueous acetic acid. The salmon calcitonin fraction from this column was adjusted to pH 6.0 with ammonium hydroxide solution. The solution is purified by ion exchange chromatography on a column filled with Whatman CM 52. Ammonia acetate buffer is used for elution. The pH of the salmon calcitonin fraction from the column was adjusted to 5 ° C with glacial acetic acid. The solution is concentrated on an ion exchange column with Sephadex G-25. 30 ml of the concentrate, removed from the column with 0.5 M sodium chloride solution, are desalted by gel filtration on a Sephadex G-25 (thin) column. The purified fraction of salmon calcitonin is freeze dried. The product is obtained as a fluffy white solid.
    Amino acid analysis (acid hydrolysis) gives the following quantities of amino acids (theoretical values are given in parentheses): Arg 1.05 (1); Asp 1.84 (2); Glu 3.14 (3); G 1 y. 3, 06 (3) 5 His 0.94 (1); Leu 5.01 (5); Lys 1.94 (2); Pro 2.11 (2); Ser 3.86 (4); Thr 5.15 (5); Tyr 0.85 (1); Val 0.95 (1).
    Cys content is not determined.
    Example 4. The procedure of Example 3 is repeated, using the same substances, in the same quantities under all district conditions, as far as practically possible, but in cycle 24 of BOC-M-tosyl-1-tyrosine, 2.5 g are replaced ( 0,008 mol) VOS-M-nitr9-1-ar.ginina. The results obtained are identical to those described in example 3.
    Example 5. The procedure of Example 3 was repeated, using the same substances, c. the same amounts and under the same conditions, but in cycle 22, instead of BOC-o-benzyl-C tyrosine, 3.45 g (0.008 mol) of C. OC-o-2-bromobenzyloxycarbonyl-1-tyrosine are taken. The results are similar to those obtained in example 3.
    Example 6 All the methods, materials and conditions are used as in Example 4, but also in cycles 18. and 11 of the Bbs-Benzyloxycarbonyl-1-leisin are replaced with 2.28 g 0.0055 mol of BOC-e. -2- chlorobenzyl-oxycarbonyl-yl-lysine. Get
    the results are the same as described
    in example 3 ,.
    权利要求:
    Claims (4)
    [1]
    1. A method for reciting cyclic peptides by closing a disulfide bridge in a non-cyclic peptide containing at least two cysteine residues, characterized in that, in order to increase the yield and simplify the process, the original non-cyclic peptide is used, one of the cysteine residues of which contains a free sulfhydryl function, and the second is the sulfhydryl function, protected by the n-alkl-thio-group, and is maintained in an aqueous solution free from oxygen at a pH of 5 to 10 in a current of inert gas. .
    [2]
    2. Method POP1, distinguishing between and and fc in that a water-alcohol solution is used.
    [3]
    3. The method according to claim 1, wherein the nitrogen is used as the inert gas.
    [4]
    4. Method POP.1, characterized in that the process is carried out at pH 7.5.
    Sources of information taken into account in the examination
    1. Schröder E., Ljubke K. Peptides,
    M., Mir, 1967, p. 350
    类似技术:
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    同族专利:
    公开号 | 公开日
    DE2540780A1|1976-04-01|
    DK409775A|1976-03-13|
    YU230075A|1982-02-28|
    JPS5156448A|1976-05-18|
    JPS5833864B2|1983-07-22|
    LU73386A1|1976-04-13|
    FR2284591B1|1978-12-01|
    AU498757B2|1979-03-22|
    BE833336A|1975-12-31|
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    DE2540780C2|1989-05-24|
    CA1045117A|1978-12-26|
    ES440922A1|1977-06-16|
    AU8473775A|1977-03-17|
    US3929758A|1975-12-30|
    NL7510761A|1976-03-16|
    YU39747B|1985-04-30|
    GB1527252A|1978-10-04|
    FR2284591A1|1976-04-09|
    NL179909C|1986-12-01|
    HU175202B|1980-06-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3560521A|1967-08-07|1971-02-02|Merck & Co Inc|Blocking groups for cysteine containing peptides|
    US3679655A|1968-06-28|1972-07-25|Hoechst Ag|N,n-disubstituted aminoethylcarbamyl protecting groups for cysteine|
    DE1950711A1|1968-10-15|1970-04-30|Ciba Geigy|New peptides and methods of making them|
    US3849388A|1968-10-15|1974-11-19|Ciba Geigy Corp|Analogues of human thyrocalcitonin|
    GB1294771A|1969-01-31|1972-11-01|Sandoz Ltd|Thyrocalcitonin derivative|
    CH523868A|1969-10-22|1972-06-15|Ciba Geigy Ag|Process for the preparation of new dotriacontapeptide amides and their derivatives|
    US3801561A|1970-10-29|1974-04-02|Sandoz Ltd|Derivatives of salmon thyrocalcitonin|
    ZA723287B|1971-06-18|1973-02-28|Hoffmann La Roche|Nonapeptides and processes for the preparation thereof|
    BE785933A|1971-07-07|1973-01-08|Ciba Geigy|PROCESS FOR PREPARING PEPTIDES CONTAINING CYSTINE|US4033940A|1975-11-12|1977-07-05|Armour Pharmaceutical Company|Cyclization of peptides|
    US4066634A|1976-08-31|1978-01-03|American Home Products Corporation|Purification of synthetic somatostatin|
    US4304692A|1978-07-24|1981-12-08|Armour Pharmaceutical Company|Synthesis of biologically active peptides|
    US4212795A|1978-11-13|1980-07-15|Armour Pharmaceutical Company|Cyclization of peptides|
    US4391747A|1982-02-12|1983-07-05|Armour Pharmaceutical Company|Des asparagine-3-calcitonin|
    US4397780A|1982-02-12|1983-08-09|Armour Pharmaceutical Company|Leucine22 -calcitonin|
    US4401593A|1982-02-12|1983-08-30|Armour Pharmaceutical Company|Glycine - 8 calcitonin|
    US4388235A|1982-02-12|1983-06-14|Armour Pharmaceutical Company|Synthesis of peptides|
    US4444681A|1982-09-24|1984-04-24|Armour Pharmaceutical Company|L-Tyrosine21 calcitonin|
    US4469632A|1982-09-24|1984-09-04|Armour Pharmaceutical Company|D-Arginine24 calcitonin|
    US4451395A|1982-11-08|1984-05-29|Armour Pharmaceutical Company|Des-serine2 -des-tyrosine22 calcitonin|
    WO1984003280A1|1983-02-15|1984-08-30|Armour Pharma|Glycine-8 calcitonin|
    WO1984003279A1|1983-02-15|1984-08-30|Armour Pharma|Des-asparagine-3 calcitonin|
    US4549986A|1983-12-23|1985-10-29|The Salk Institute For Biological Studies|Human CGRP|
    US4623716A|1984-11-01|1986-11-18|Usv Pharmaceutical Corp.|Process for the preparation and purification of peptides|
    JPH0517511Y2|1985-09-30|1993-05-11|
    US4644054A|1985-10-01|1987-02-17|Kempe Tomas G|Calcitonin analogs with amino acid substituents at position 31|
    US4658014A|1985-12-20|1987-04-14|Kempe Tomas G|Synthetic peptides with calcitonin-like activity|
    US4687839A|1985-12-23|1987-08-18|Kempe Tomas G|Calcitonin gene related peptide analogs with C-terminal D-amino acid substituents|
    US4697002A|1985-12-23|1987-09-29|Kempe Tomas G|Calcitonin gene related peptide analogs with amino acid subdstituents at the penultimate position 36|
    US4820804A|1987-04-21|1989-04-11|Rorer Pharmaceutical Corporation|Analogs of [1,7-di-alanine, des-19-leucine]calcitonin|
    US4824936A|1987-05-06|1989-04-25|Rorer Pharmaceutical Corporation| calcitonin|
    JP2682061B2|1988-10-07|1997-11-26|藤沢薬品工業株式会社|Method for forming intramolecular disulfide bond of peptide|
    ES2051647B1|1992-12-10|1995-01-16|Lipotec Sa|PROCEDURE FOR THE PREPARATION OF CALCITONINA DE SALMON.|
    RU2135204C1|1993-06-29|1999-08-27|Ферринг Б.В.|Compositions for nasal administration of desmopressin|
    AT193892T|1993-06-29|2000-06-15|Ferring Bv|SYNTHESIS CYCLIC PEPTIDE|
    US5482931A|1993-06-29|1996-01-09|Ferring Ab|Stabilized pharmaceutical peptide compositions|
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    法律状态:
    优先权:
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