JPS60500054A - des-asparagine-3-calcitonin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Des-asparagine-6-calcitonin Field of the invention The present invention provides biologically active calcitonin and biologically active calcitonin. Peptides convertible to calcitonin and methods for preparing such peptides and calcitonin O regarding Background of the invention All known natural calcitonins contain a chain of 32 amino acids.

For example, salmon calcitonin has the formula:

H2 U.S. Pat. No. 3.926.938, U.S. Pat. No. 4.062.815.

3.929,758, 4.033.940 and 4.217,2 No. 68 discloses an improved method for synthesizing calcitonins, including the above-mentioned salmon calcitonin. Disclosed.

Natural calcitonin includes salmon calcitonin, cow, and pig.

There are sheep and eel calcitonins and human calcitonins. These are all It has asparagine at the 3rd position.

Outline of the invention The inventors discovered that the 31-amino acid peptide has more biological activity than its known homologous calcitonin. discovered the synthetic calcitonin R peptide. The remarkable structural difference is that the inventor of wood The amino acid chain of the new Hefty P, such as No asparagine residue is contained in the position. Synthetic peptides Using the IUPAC4UB nomenclature for degeneration, this novel des-X) calcitonin (where X is asparagine). [Bioche m-Biophys-Acta, Male 1-5 (1967)) This new haftide It has good efficacy and quality compared to known calcitonin.

Detailed description of the invention In general, wood inventors use a solid-phase synthesis method, using benzuhito-9lylamine resin ( Starting from a resin called BHA resin). This resin is made of styrene and divinyl vinyl. derived from cross-linked polystyrene bead resin produced by copolymerization with . This type of resin is known, and its method of preparation is described in detail by Binitsuta et al. [P ietta, P-3, and Marshall, G.R., Chem-Co. mmun, /) 50 (1970)] This crosslinked polystyrene BHA resin is Available from chemical suppliers. To represent this BHA resin, in the following formula: ■ is the polystyrene part of the resin.

In this synthesis, the insoluble resin is added to the insoluble resin at one time until the whole takuputide reren lead is formed on the resin. Add 1 amino 0.

Amino acid functional groups are protected with blocking groups. The α-amino group of amino acid is 3 protected with a butyloxycarbonyl group or its equivalent. This α-6-butyl The oxycarbonyl group is called BOG. The hydroxyl groups of serine and threonine are benzene or benzyl-derived groups such as 4-methoxybenzyl, 4-methylbenzyl, 3 ．． 4-dimethylbenzyl, 4-chlorobenzyl, 2.6-0 chlorobenzyl.

Protected by 4-nitrobenzyl, benzhito9lyl or equivalents thereof.

The term BZ is used to represent benzyl or benzyl-derived groups.

The hydroxyl group of tyrosine is either unprotected or the benzyl group described as BZ group in the previous section. or protected with a benzyl-derived group, or indyloxycarbonyl or Benzyloxycarbonyl derivative 1 For example, 2-chlorobenzyloxycarbonyl or protected with a 2-bromobenzyloxycarbonyl group or their equivalents. It will be done. No protecting group, BZ group, benzyloxycarbonyl group or benzyloxycarbonyl group The term W is used to represent a rubonyl derived group.

The thiol group of cysteine is the benzyl or benzyl-protected group represented by BZ above. or n-alkylthio groups such as methylthio, ethylthio, n-propylthio , n-butylthio or their equivalents.

When W No. R2 is BZ to represent n-alkylthio group or BZ, n-a He R is used to represent rukylthio. Others + R, is another cysteine In this case, R2 is BZ. The guanidine group of arginine is Protected with nitro groups, tosyl groups or their equivalents. Nitro group or tosyl group The symbol T is used to represent. The ε-amino group of lysine is a benzyloxycarboxylic group. Bonyl group or 2-chlorobenzyloxycarbonyl, 3,4-dimethylbenzyl Indyloxycarbonyl derivatives such as oxycarbonyl or their equivalents more protected. benzyloxycarbonyl group or benzyloxycarbonyl Use the busy symbol V to represent a derivatized group. used on imidazole nitrogen of histidine The protecting group used is a benzyloxycarboxylic group such as the one named V mentioned above for lysine. Bonyl group and benzyloxycarbonyl derivatives. γ-cal of glutamic acid The bonic acid group is a benzene group such as that described for protecting the hydroxyl groups of serine and threonine. Protected by a zyl or benzyl derived group.

These protecting groups are represented by the symbol BZ. .

The inventors' novel des-ASn- has the same activity as known salmon calcitonin. The formula for salmon calcitonin is written as follows.

5 As can be seen from the above formula, 61 amino acids are involved, and the positions in one formula are Numbered according to generally accepted methods. i.e. on one end of the chain Starting at position 1 of CYS, position 3 of the asparagine missing from the chain is omitted 12. @ Ends at position 32 PRO at the other end.

To make the explanation less confusing, the same numbering system is used for the cycle of 1 synthesis. Follow the system. The assembly of amino acids consists of cycle 32 involving the coupling of proline. starting with cycle 61 for the coupling of threonine, and so on. be.

Preferred amino acid reactants used in each of the 32 synthetic cycles are listed in Table 1 below. Ru.

32 BOC-L-proline 31 BOG-〇Menjiru L-) Leonine 30 BOG-Glycine 29, BOC-〇-benzy-L-serine 28, BOC-glycine 27 BOC-0-benzyl-L-threonine 26 BOC-L-asparagine p-nitrophenyl ester 25 Boo-0-benzyl-L-threonine 24 BOC-Ω-Nido I: IL-7/I/Ginine 0rBOC-〇-Tosyl L-Arginine 23 BOG-L-Pro IJn 22 BOC-0-Indyl-L-Tyrosine L BOC-L-Syrosine or B OC-0-2 Benzyloxycarbonyl bromide-L-tyrosine 21 BO(,-0 -benzyl-L-) leonine 20 BOC-L + glutamine p-nitropheny ester 19 BOC-L-leucine 18 BOC-E-CBZ-L-1)) 51JtBOC-ε-2-pendichloride oxycarbonyl-L-lysine 17 BOC-N(im)-GBZ-L-hyx Tisine 16 BOC, L-leucine 15 BOC-L-glutamic acid γ-benzyl ester 14 BOC-L-/ y, J Min p-nitrophenyl ester 13 BOC-L benzyl-L- Serine 12 BOC-L-leucine 11 BOC-E-CBZ-L-IJ Jinmataha BOC-ε-2-Penzylochloride Xycarbonyl-L-lysine 10, BOG-glycine 9 BOC-L-leucine 8, BOC-L-valine 7 BOG-3-ethylthio-L-cystiy, BOC-8-methylthiome- Cysti 4 BOCi-3-n-propylthio-cysline or BOG- 3-n-butylthio-L-cysteine 6 Boc-o-benzyl-L =, ) leonine 5 BOC-0-benzyl-L-serine 4 BOC-L-leucine 2 BOC-〇-Benzyru-L-serine I -L-Sistine.

BOC-6-benzyl-L-cysteine or BOC-3-3,4-dimethylbenzene Njiru-shi-systemen.

Bisou BOC-L-cystine Each amino acid derivative noted in Table I is commercially available.

cycle 32 Coupling of proline to BHA resin Used in all steps of peptide synthesis The reactor is equipped with a feedstock addition inlet at the top to filter the soluble reaction mixture and wash solvent. ・It is a glass container equipped with a sintered glass plate at the bottom for removal. Filtration is vacuum or by using nitrogen pressure. Shake the entire container and remove the contents of the container. Or stirred with a mechanical stirring device.

In cycle 62, the BHA resin is placed in the reactor and the methylene chloride, chloroform 9° of dimethylformamide is added to the resin in a solvent such as benzene or their equivalents. Suspend in the solvent at a ratio of about 6 to 12 parts solvent per duram.

This is combined with about 1 to 6 equivalents of BO (L-propylene) per free amine equivalent of the BHA resin used. Add Lorin. After 5-10 minutes qn-:) cyclohexylcarbodiimide Coupling agents such as DCC) (CQ or other diimide coupling agents) are used. The amount of diimide coating 1) used is based on the amount of BOC-L-proline used. The amount is 0.5 to 2.0 equivalents.

BOC-L-proline has active ester derivatives, azide derivatives, and pairs thereof. When using a nominal anhydride derivative or an appropriately selected anhydride derivative, the Coupling is possible even without a primer. The active ester derivatives that can be used are 2 -nitrophenyl ester, 4-nitrophenyl ester, hantafluorphe Nyl ester + N-hydroxysuccinimide ester or their equivalents. Ru. The amount of active ester used is 1 to 10 equivalents per equivalent of free amine of BHA resin. It is.・BHA resin, bath agent, BOC-L-proline and coupling agent or A reaction mixture consisting of BOG-L-proline active ester was added to the test sample of ninhydride. Phosphorus test [E, Kaiser, etc. + Anal, Bioche Stir mechanically until m, 34.595-8 (1970) 1 indicates completion of the reaction. Or shake.

After the coupling reaction is complete, methylene chloride, chloroform, methyl alcohol, BOC-L-proline resin with a solvent such as chlorine, dimethylformamide P or acetic acid. Wash. The amount of cleaning solvent ranges from 5 to 20 parts per gram of BHA resin initially used. LII solvents are suitable. Wash if you want to stop the coupling reaction before it is completed. The remaining free amino groups on the BOG-L-proline resin are removed by removing the excess Acechi 9 It is preserved from further reactions by acetylation with a binding agent. acetylating agent BOC-L-pro-11 resin with acetylating agent solution for 05 to 12 hours This is done by stirring for a period of time. N-acetylimidazole/methylene chloride solution Acetylating agents such as liquid or acetic anhydride and triethylamine/chloroform mixtures are used. used. The amount of acetylating agent used is based on the free amine titer of the starting BHA resin. The amount is 0.5 to 5.0 equivalents.

The coupling reaction for producing BOC-L-proline resin is described by the following formula: described.

Removal of protecting groups from BOC-L-proline resin BOC-L-proline resin prepared as above. The Lorin resin was washed with a solvent such as those described above, and then mixed with methylene chloride and chloroform. Mixing trifluoroacetic acid (TFA) with a solvent such as alcohol, banzene, or their equivalents. Protecting groups are removed by co-stirring with reagents such as substances. The amount of TFA in the solvent is 1 It varies from 0% to 100%. The amount of TFA-solvent mixture is equal to the amount of BHA initially used. It varies from 3 to 20 m1 per resin duram. Reaction time is approximately 10 minutes to 4 It's time.

Protecting group removal is stopped by filtering and removing the TFA-solvent mixture. . Residual TFA may be methylene chloride, chloroform, benzene or their equivalents, etc. A solution of 5 to 60% triethylamine dissolved in the solvent of BHA resin Durum removed from the L-pro 11 resin by washing with 3 to 207 d. It will be done. Other tertiary or secondary organic amines, such as triethylamine, can be used instead of triethylamine. Methylamine, N-ethylbi, lysine-diisopropylamine or their equivalents You can also use objects. The free amine titer of L-proline resin is My (Dorman) titration method ()'' - Mann, L., C.

Tetrahedron Letters,’ 1969.2319-21 ) is measured. The protecting group removal reaction is described by the following formula.

cycle 31 Pro1) BHA resin obtained as a result of cycle 32 as coupling solvent BOC-0-BZ-L) leonine derivative was added and the mixture was similarly balanced. Reach a state of equilibrium. Add the coupling agent DCC and add ninhydride 1 After the reaction mixture test showed the reaction to be complete, the reaction mixture was transferred to the BOC-0-BZ) level by E-filtration. Separate from onylprolyl BHA resin. Cleaning the petite resin with a solvent . Amounts of reactants and solvent as well as reaction times were similar to those described for cycle 62. be. The BOC group is removed from this sputide tree by the protecting group removal method described in Cycle 62. Remove from fat. Subsequently obtained 0-BZ-threonylpro II BHA resin+ -i The various reactions of cycle 31 provided for cycle 60 are shown by the following formula.

For convenience, the resulting resin is referred to as peptide using the following abbreviations.

In cycle 30, BOC-glycine was substituted for BOG-0-BZ-)lireonine. The coupling reaction and protecting group removal were carried out in the same manner as in cycle 31, except that Perform the elimination reaction. Coupling reactions and retention group reactions are written as follows.

In cycle 29, the amino acid derivative is replaced with BOG-・0-BZ-L-serine. Coupling and protecting group removal reactions are performed as in cycle 31, except that . This is written as follows.

13 cycle 28 In cycle 28 (・engineering, except replacing the amino acid reactant with BOC-glycine) , coupling and protecting group reactions as described in cycle 61. these The coupling and protecting group removal reaction is written as follows.

cycle 27 The coupling and protecting group removal reactions in this cycle are the same amino acids as in cycle 61. Using the reactants, the following compound is obtained.

cycle 26 The coupling reaction in cycle 26 consists of BO (the active ester of 3-L-asparagine). This is done using a tel derivative. Active ester method is an alternative to DOC coupling agent used with BoC-asparagine or BOC-glutamine.

The reaction involves converting the active ester derivative of BOC-L-asparagine to the liberated BHA resin. Use 2 to 10 equivalents per equivalent of amine, 9. dimethylform A mixture of Muamide 9 and benzene, methylene chloride or chloroform, or a mixture thereof 2 to 20 ml of solution per duram of BHA resin initially used in the IC. It is done in a dosage amount.

The BOC was filtered from the polymer resin when the nitrogen test showed that the reaction was complete. passed and removed. Active ester derivative used i12-nitrophenyl ester fluorophenyl, 4-nitrophenyl ester, fluorophenyl or their equivalents It is. The active ester portion of the derivative is designated as AE. The coupling reaction is as follows wake up.

The protecting group removal reaction to remove the BOC group is performed as in cycle 62.

Cycle 25-21 For each cycle, cycles 25 through 21, the method and reactant proportions are the same as cycle 31. Similarly, BOC-BZ-L-) leonine was added in cycle 25 and BOC-BZ-L-) leonine was added in cycle 24. BOC-ω-T-L-arginine in cycle 23 and BOG-L-prolyl in cycle 23. in cycle 22, BOC-W-L-tyrosine in cycle 21, and BOC-W-L-tyrosine in cycle 21. -0-BZ-L-) leonine. obtained after cycling photons The compound is written as follows.

5 cycle 20 In cycle 20, BOO-L-glutamine active ester is used as an amino acid derivative. Coupling using the same method and reactant proportions as in Cycle 26 using derivatives. and performing a protecting group removal reaction. The produced compounds are as follows.

In cycle 19, BO (3-L-leucine was used as the amino acid derivative and The reaction was carried out in the same manner as in Cycle 31. The product compounds of cycle 19 are as follows: be.

cycle 18 Cycle 18 uses the amino acid derivative BOC-ε-V-I, -lysine. Ru. Otherwise the method of cycle 18 is similar to cycle 61 and is described below. A product is obtained.

Cycle 17-15 Cycles 17 to 15 are BOC-N(im)-V-L-hi in cycle 17. Stidine and BOC-L-leucine as reactant in cycle 16, cycle In No. 15, BOC-L-glutamic acid BZ ester (BZ is serine and and threonine). Cycle 15 gives the following compound.

Cycle 14-8 Cycle 14 uses BOC-L-glutamine-AE as the amino acid derivative. , is performed in the same manner as in cycle 20.

Cycles 16 to 8 are 'rike #13 Teha BO (3-0-BZ-L-Seri and BOC-L-leucine in cycle 12.

BOC-ε-V-L-lysine in cycle 11 and BOC-group in cycle 10. lysine in cycle 9, BOC-L-leucine in cycle 8, and BOC-L-leucine in cycle 8. -17 using valine Similar to cycle 61 except that the following compound is obtained.

Cycle 7 uses BOC-8-L-cysteine as the amino acid derivative. The process is similar to cycle 61, except that: The compound written as the result of cycle 7 has the following formula In the formula, R2 is an alkylthio or BZ group.

Cycle 6-4 and 2 Cycles 6 to 4 contain BOC-0-BZ-L in cycle 6 as an amino derivative. -threonine in cycle 5 and BOC-BZ-L-serine in cycle 4. BOC-L-leucine was used in cycle 2, and BOC-BL-L serine was used in cycle 2. The same process as in cycle 61 is performed except that. The compound obtained from cycle 2 has the following formula It is as follows.

This cycle is repeated as cycle 7 using BOC-8-R-L-cysteine derivative. The same is done. The R group selected for cysteine is used in cycle 7. It can be the same as or different from what is available. For example, select for cycle 7 If the derivative to be treated is BOG-S-ethylthio-L-cysteine, then the cycle The derivative of Group 1 is BOC-8-4-methoxybenzyl-4-cysteine, BOC-8-4-methoxybenzyl-L-methoxybenzyl-L as cycle 7 - If cysteine is selected, this derivative is also used in cycle 1. sa The compound obtained from cycle 1 is represented by the following formula.

9 However, in the formula, R□ is 5-n-alkyl-CYS or BZ, and R2 is 5-n-alkyl-CYS or BZ. Kyl or BZ, R□ is 5-n-alkyl if R2 is BZ, and R2 is R'IJ''-5-n-alkyl or CYStc is BZ.

Cycle 1 represents the completion of the resin peptide and the resin peptide is removed from the reactor. , dried in vacuum. The weight of resin R buti 1 is the BHA resin initially used in the synthesis. It is expected to be 2.0 to 4.0 times the weight.

Resin R buty r cleavage The resin obtained from cycle 1 is liquid hydrogen sulfide (HF). It is treated with and cleaved. HF cleavage is performed on resin heftide and anisole (resin peptide). 0.5 to 5 ml' for each gram of liquid HF (resin is 0.5 to 5 ml') Treat at -20°C to +15°C for 0.5 to 20 hours at 2 to 20°C per gram. It is done with understanding. After the reaction, excess HF is removed by evaporation, and the remaining peptide and resin binder are removed. Mixtures of organic acids such as ethyl acetate, :) ethyl ether, benzene or similar Extract with solvent to remove anisole and HF residues. Peptides in vinegar, acid aqueous solution It is separated from the resin beads by extraction. Vepti 1 at this stage is circular Instead, a cyclic disulfide bond is formed between cysteine at the 1st and 7th positions in one molecule. It is a non-cyclic product.

HF treatment removes the S-alkaline group on the thiol group of the cysteine residue at the 1st or 7th position. All blocking groups are removed from the Zuptide except the killthio blocking group. Ru.

5-n-alkylthio-L-cysteine residues are stable to HF cleavage operations; During the cleavage and extraction operations, it remains as is. 5-BZ-L-cysteine is HF Cleavage gives a cysteine residue with a free thiol group. Synthesis of wood inventors etc. In this case, both types of blocking groups are used in combination with each other at the 1st position and the thickness 7 position. did.

One of six types, depending on the blocking group selected for.

BOC-3-BZ-L-cysteine derivative used in resin peptide synthesis cycle 1 and BOC-8-n-alkylthio-L-cysteine was used in cycle 7. If C, the peptide obtained after HF cleavage will be of type I, with a free titanium at the first position. has an all group and a 5-n-alkylthio group on the cysteine residue at the 7th position. will have. The inventors call this type I o buti 1, and it is expressed as a linear equation. Table■ H-CYS-3ER-LEU-3ER-THR-CYS-vAL-LEU-GL U-LYS-21 LEU-8ER-GLN-GLU-LEU-HIS, -LYS-LEU-GLN +THR-TYR-PRO-ARG-THR-ASN-THFi-GLY-S ER, -GLY -THR-PRO-NH2 Conversely, in cycle 1 BOG-3-n -alkylthio-L-cysteine derivative, BOC-8-BZ at the 7th position If -L-cysteine was used, the peptide F obtained from the cleavage would be of type ■. It will be expressed by a linear equation.

LEU-3ER-(,LN　-GLU-LEU-Hl5-LYS-LEU-GL N-THR-TYR-PRO-ARG-THR-ASN-THR-GLy-5E R-GLY-THR = PRO-NH2 Protecting group 5-n-alkyl at the first position In contrast, wood inventors use a cysteine group (this cysteine group is form a cystine group with cysteine), in this case the cysteine at the 7th position is retained. The BZ group is used for protection. Bis-BOG-L-Si as reactant in cycle 1 cycle 7/) using BOO-8-BZ-L-system as the reactant. If a peptide is used, the peptide obtained from the cleavage will be of type ■ and in the linear equation expressed.

-GYS LEU-8ER-GLNL-GLU-L;EU-HIS-LYS-LEU-(, LN -THR-TYR-PRO-px, -THR-ASN-THR-GLY- SER-GLY-Tm-PRO-NH2 Both 1st and 7th positions are BOC-BZ- If L-cysteine was used as a reactant, the peptide resulting from the cleavage would be Ip■, which is expressed by the following formula.

H-CYS-8ER-LEU-8IiR-T) (R-CYS-VAL-LEU- GLY-LYS, -LEU-8 original t-GLN-GLu-bEu-H engineering s-r, y s-jEu-Gt, N-THR-TyR-PRO-ARG-THR, ASN-T HR-GLY-5ER-GLY-THR-PRO-NH2 Type I, If and m <Conversion of peptides to cyclic disulfide peptides involves the conversion of crude peptides from HF cleavage. The aqueous acetic acid solution is distilled with water until the final volume is cleaved. This is done by diluting it to 200 grams. The pH of this solution is ammonium hydroxide. 5 to 10 by the addition of monium solution, and the mixture is heated with an inert gas such as nitrogen. Stir in a sealed container for about 2 to 48 hours. The reaction occurs when the off-gas stream It can be stopped when it no longer contains rukymercaptan. by adding glacial acetic acid The pH of the reaction mixture is lowered to about 6.5-5.5.

Conversion of type IV s peptide to cyclic disulfide h-J peptide 9 oxidize to form a ring structure containing cysteine at the 1st and 7th positions within the ring. It is done by known classical methods.

The intermediate takuputide is type 1. If it is either Il, m or ■, the third position a An amino acid corresponding to any known calcitonin, except that sparagine is omitted. The amino acid chain Veptyl can be synthesized, and when the peptide synthesized in this way is purified, , was found to have the same type of biological activity as this known calcitonin.

Calcitonin thus synthesized is named des-Asn-calcitonin.

This follows IUPAC-IUB nomenclature.

3 Purification of crude desAsn SOT The phase isotide solution at pH 5.0 synthesized above was concentrated using the ion exchange resin method. Shrink. This concentrate was processed using a combination of gel filtration and ion exchange chromatography. Purify by method. The final purified product is produced as a fluffy white solid by freeze-drying from the solution. obtained as. Amino acid analysis of this product accurately matches the desired peptide do.

Below is a specific example of peptide preparation.

Example 1 Resin activation BHA resin [5 g] with an amine titer of 0.61 meq/g was added to a Spartz matrix. Shwarz-Man, Inc.: x-Orangepurg, York ) was placed in the reaction vessel of a peptide synthesizer manufactured by A. Add this resin to 25 g of the following solvent. and filtered after each treatment.

Treated with chloroform for 2 minutes twice.

10% triethylamine/chloroform treatment for 5 minutes, twice chloroform treatment for 2 minutes. Reason Methylene chloride treatment for 2 minutes 6 times cycle 32 Coupling: the BHA resin, methylene chloride 25g and Boc-r,-proline 2.5'8.9 (0,012 mol) was stirred for 10 minutes. Dicyclohexinka 12.0 d of methylene chloride solution of rubodiimide (1 rrL of solution corresponds to 11 DCC) milliequivalent) was added to the reactor and the mixture was stirred for 6 hours. Filter the reaction mixture Remove from the reactor and continuously wash the BOG-Prolyl BHA resin with the following solvents: 25 m/-2 minutes) The washing liquid was filtered and removed each time.

Methylene chloride 2 times Methyl alcohol 2 times Methylene chloride 3 times After acetylation, the resin was treated with 1.57 LA of triethylamine (TEA) and acetic anhydride. 1m7! The mixture was stirred for 2 hours with a mixture of chloroform and chloroform. reaction mixture k f was filtered and removed, and the resin was subjected to the following washing (2 minutes - 25ml).

Chloroform twice Methyl alcohol 2 times Methylene chloride 6 times Protecting group removal: BOC protected resin trifluoroacetic acid (TFA) 15d and chloride Stirred for 5 minutes with a mixture of 15 ml of tyrene. This mixture is filtered and removed, and Fat.

Stirred for 60 minutes with a second mixture of 15ml of TFA and 15ml of methylene chloride.

The reaction mixture was filtered and removed, and the resin was subjected to the following washing (25d,).

Methylene chloride twice, 2 minutes each Methyl alcohol twice, 2 minutes each Chloroform twice, 2 minutes each 10% TEA/Chloroform twice, 10 minutes each Chloroform twice, 2 minutes each Methylene chloride twice, 2 minutes each T, J　-7' Titrate the loline BHAi fat to measure the amount of amine or proline. Established. This value is 0.55 meq of amine or proline per duram of resin. Ta.

25 Go and BOC-0-benzyl-L-) leonine 6.49 (0,011 mol) was stirred for 10 minutes. Next, dicyclohexylcarbodiimide 9 was dissolved in methylene chloride. 11.0 ml of solution (DCCI 1 milliequivalent per 1 d of solution) (DGGI total 0.011 r-ru) was added to the reactor and the mixture was stirred for 2 hours. Reaction mixture from reactor The resin was washed continuously for 2 minutes with 25 grams of the following solvent, and the washing solution was filtered and washed each time. Removed.

Methylene chloride 2 times Methyl alcohol 2 times Methylene chloride 3 times A ninhydrin test was negative.

Protecting Group Removal: Also for this cycle, the protecting group removal method described in Cycle 52 is used. repeated.

Cycles 30 to 27 The coupling and protecting group removal methods used in these cycles are based on threonine derivatives. Cycle 31 was the same as cycle 31, except that the following amino acid derivatives were used instead of .

Cycle 30...BOC glycine 1.92.9 (0,011 mol) cycle 29...BOC-0-benzyl-L-serine 3.25g (0,011 mol) Cycle 28...using the same reactants as in cycle 60 Cycle 27...cycle Cycle 26 using the same reactants as in 31 Coupling: Peptide resin dimethylformamide obtained from cycle 27 '(DMF) 257714 each. Next, the resin was mixed with BO in DMF35. C-L-asparagine p-nitrophenyl ester 5.82.9 (0,016 5 mol) solution for 24 hours. FA the reaction mixture.

The resin was washed successively twice with 25 mA' each of the following solvents for 2 minutes. DMF , methylene chloride/methanol, methylene chloride. Each solvent is filtered and removed, Dolin test was negative.

Protecting group removal: The protecting group removal method used in cycle 32 was repeated.

cycle 25 Coupling and protecting group removal were performed in a similar manner using the same reactants and amounts as in cycle 31. It was done by method.

cycle 24 Coupling: Resin peptide obtained from cycle 25 in DMF each 251 Lt. Washed twice in succession. Next, apply this resin haft 4 to Boc-N-y-)silyl L. - 1 with a mixture of 7.06 arginine (0,0165 mol) and 25 mol of DMF Stirred for 0 minutes. Next, DCCI16.5rd (DC (equivalent to 0.0165 mol of CI) was added and the mixture was stirred for 6 hours. reaction mixture The material was filtered and removed. This resin was applied twice in a row with each of the following solvents. Washed for minutes. DMF, methylene chloride, methyl 7/l/=r-l, chloride Methylene. The human e+l test was negative.

Removal of the protective group used in cycle filtration 2 was repeated.

cycle 26 Coupling: The peptide resin obtained from cycle 24 was 3.54 g (0, [) 165 mol) and 2571 LI of methylene chloride! ! with Stir for 10 minutes and add 16.5 ml of methylene chloride (17) DCCI (D CCI (corresponding to 0.0165 mol) was added and the mixture was stirred for 6 hours. reaction mixture The compound was filtered and removed, and the resin was washed twice with 25 grams of each of the following solvents. Ta. Methylene chloride, methyl alcohol, methylene chloride. Each washing solution is filtered and removed. Ta. A ninhydrin test was negative.

Protecting group removal: The protecting group removal method used in cycle 62 was repeated.

Cycles 22 and 21 The coupling and protecting group removal methods used in these cycles were Except for the point 7 in which the following amino acid derivatives were used instead of BOC-L-pro 1). It was similar to cycle 23.

Cycle 22...BO (3-W-L-tyrosine 8.1.5g (0,01755 mole) Cycle 21...BOC-○-benzy-L-threonine (0, [11 65 moles) 8 cycle 20 This method uses BOC-L-glutamine p-nitroph instead of asparagine derivatives. cycle except that 6.509 (0.0165 moles) of phenyl ester was used. It was the same as 26〇 Cycles 19 to 15 The following amino acid derivatives were used instead of threonine induction! Except cycle 61 A similar method was used.

Cycle 19...BOC-L-leucine 2.54 g (0,011 mol) cycle Chlor 18...BOG-ε-2-chlorocarbobenzyloxy-L-lysine 45 5. ! i' (0,011 mol) Cycle 17...B'OC-N(im)-ka Lebo-benzyloxy-L-histidine Cycle 16...See cycle 19 Cycle 15...BOC-L-G Rutamic acid-γ-benzyl ester 6.70 (0,011 mol) cycle 14 Same as cycle 20 cycle 16 In the coupling reaction, instead of the proline derivative, BOC-O-vanjiru L-Se1 Same as Sukokuru 26 except that 4.86 g (0,0165 mol) of 1-ton was used. Various methods were used.

Cycles 12-9 Except that the following amino acids were used instead of threonine derivatives in the coupling reaction. Ki-cycle 61 and similar person 29 The law was used.

Cycle 12...Same reactants used in cycle 19.

Cycle 11...Reactants were the same as Cycle 18.

Cycle 10: Same reactants used in cycle 60.

Cycle 9: Same reactants as used in cycle 19.

cycle 8 Coupling: Resin Iptidowo from Cycle 9.

BOC-L-valine 3.85 g (0.Q165 mol) and methylene chloride 25 g and stirred for 10 minutes. Next, 16.5ml of DOCd in methylene chloride! (D CCI (corresponding to 0.0165 mol) was added and the mixture was stirred for 16 hours. reaction The mixture was filtered and removed. The resin peptide was applied twice in succession with each of the following solvents. Washed for 2 minutes. Methylene chloride, methyl alcohol, methylene chloride. The washing liquid It was filtered and removed each time.

Protecting Group Removal: See Cycle 31 Cycle 7 In the coupling reaction, BOC-8-ethylthio-L- Cycle 6 except that cysteine 3.09 Il (0,011 mol) was used. A method similar to 1 was used.

Cycool 6 The reactants and methods used were similar to Cycle 31.

cycle 5 The reactants and methods used were similar to cycle 29.

The reactants and methods used were similar to cycle 19.

cycle 2 The reactants and methods used were similar to cycle 29.

cycle 1 The reactants and methods used included BOC-8-1)-methoxy instead of the threonine derivative. except that 3.75 g (0,011 mol) of cybenzyl-L-cysteine was used. , was similar to cycle 61.

After cycle 1 is completed, the resin peptides are continued twice with 251 L11 of n-hexane each. and washed. The k-butyl-r substance was taken out of the reactor and kept at 40°C in an electric vacuum dryer. , 0.1 m) and dried for 24 hours.

Cleavage with hydrogen fluoride Dry resin Ipti 1-'' (2 g) and 2 Nl of anisole were placed in a Teflon reaction vessel. It was placed in Place the container equipped with a Teflon-coated magnetic stirrer into 1 ice cube. - placed in an acetone bath and hydrogen fluoride gas 10 - was condensed into the vessel. this The mixture was stirred in a water bath at 0° C. for 1 hour.

Hydrogen fluoride was removed by evaporation under reduced pressure. The residue was mixed with 101 ml of ethyl acetate for 6 There was a mouth frame. 0.11 from resin beads The peptides were extracted with 120 d of molar acetic acid aqueous solution.

Cyclization of peptides Distilled water was added to the acetic acid aqueous extract obtained from hydrogen fluoride cleavage to give 0 ．． Diluted to 2 liters. Add concentrated ammonium hydroxide to bring the pH of the solution to 15. Adjusted to.

The solution was stirred in a sealed container under nitrogen flow for 24 hours.

At this point, no ethylmercazotane was detectable in the output nitrogen stream. nitrogen flow Ethyl mercaptan content number - The stream was dissolved in Nillman's reagent Ellman.

G, L,) Arch, Biochem, Biophys, * 82.7O-7 (1969)]. Addition of glacial acetic acid to the reaction mixture The pH was adjusted to 5.0.

Purification of crude des-Asn-3CT 0.2 liters of the pH 5.0 solution obtained in the above synthesis was added to Concentration was performed using a (Sephadex) 0-25 ion-exchange column. 0.7 molar sodium chloride 1) The concentrate 25- removed from the column with the rum solution 8 salt-free and passed through Sephadex G-25 (fine grain) gel filtration column to 0.06 It was purified by elution with a 6 molar aqueous acetic acid solution. death from this column -pHYs of ASN3-8CT fraction 6.0 by addition of ammonium hydroxide bath solution Adjusted to. This solution was applied to a Whatman 0M52 column. Elute with ammonium acetate buffer using ion exchange chromatography. Purified. pHy of Desu ASN3-8OT fraction from this column, rice vinegar 2 The temperature was adjusted to 5.0 by adding a supplement. This solution was added to SP-Sephadex C-25 It was concentrated using an on-exchange column.

Concentrate 30 removed from the column with 0.7 molar sodium chloride solution m/ was desalted using a Sephadex G-25 (fine grain) gel filtration column. Refining is The chloride fraction was collected and freeze-dried. The product was obtained as a fluffy white solid. It was. The ratio of the product based on amino acid analysis is as follows, with the theoretical value in parentheses. write down

Aspl, 0 (1) - Thr4.9 (5), 5er3.8 (4), G ln3.1 (3)-Pro2.1 (2), Gly3.0 (3), Leu4. 9(51HisO, 9(1).

Tyro, 85 (11Cysl, 9(2), Lysl, 9(2), Argo, 9(1) This product assayed 4300 MRC units per dab.

Although only some uninvented embodiments have been described in detail, those skilled in the art will be able to understand one invention. It is understood that many other specific embodiments may be made within the spirit and scope of the claims. It will be clear that many variations are possible.

The formula of the new des-Aqn-eel calcitonin of the inventors is written as follows: Ru.

Cys-8er-Leu-8er-Thr-Cys-Val-Leu-Gly -Lys-LeuSe r -Gln-Glu-Leu-Hi 5-Lys-L e u-Gln-Thr-Tyr-Pr.

Arg-Tnr-Asp-Val-Gly-Ala-Gly-Thr-Pro- NH2 The present invention uses des-Asn homologues of bovine, porcine and ovine calcitonin. It also includes.

Procedural amendment Mr. Kazuo Wakasugi, Commissioner of the Patent Office 1.Display of the incident Showa Patent Application No. PCT/US8ro10[]220 2. Name of the invention des-asparagine-6-calcitonin-6, corrector Relationship to the incident: Patent applicant 6. Contents of amendment The statement will be amended as follows.

LEU-GLU-LYS- THR-CYS-VAL-LEU-GLY-H-CYS-8ER-LEU-8E R-THR-CYS-VAL'-LEU-GLY-LYS-and above international search report

Claims (1)

[Claims] 1) Des-X3-calcitonin, where X is asparagine. 2) Calcitonin is that of salmon, cow, pig-sheep or eel' = Aogu Calcitonin according to item 1. 3) A peptide with the following structure. Peptide 9゜ having the structure A peptide with the following structure. However, R□ is 5-n-alkyl, cys, or E, and R2 is 5-n-alkyl. and when R2 is H, Ro is 5-n-alkyl, Cys or H. and R1 is H, R2 is 5-n-alkyl or H, and Ro is 5- In the case of n-alkyl or qs, R2 is H and the 5-n-alkyl group is methyl From thio, ethylthio, n-propylthio, n-butylthio and their equivalents selected from the group. It has a structure similar to that of petido. However, R is 5-n-alkyl-Cys or H, and R2 is 5-n-alkyl or H, and when R2 is H, R□ is 5-n-alkyl-Gys or H And when R, KaH, R2 is 5-n-alkyl or H, and -R, is 5-n- In the case of alkyl or CyS, R is H and the 5-n-alkyl group is methylthio , ethylthio, n-propylthio, n-butylthio and their equivalents. selected from a group. 1