CA1149378A - Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh - Google Patents
Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-ohInfo
- Publication number
- CA1149378A CA1149378A CA000406187A CA406187A CA1149378A CA 1149378 A CA1149378 A CA 1149378A CA 000406187 A CA000406187 A CA 000406187A CA 406187 A CA406187 A CA 406187A CA 1149378 A CA1149378 A CA 1149378A
- Authority
- CA
- Canada
- Prior art keywords
- dnp
- glu
- dcc
- added
- hoobt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
PROCESS FOR THE MANUFACTURE OF LH-RH AND LH-RH ANALOGS
USING ?Glu-His(DnP)-OH
Abstract of the disclosure:
Process for the manufacture of LH-RH andLH-RH ana-logs, which comprises reacting ?Glu-His(DnP)-OH with the corresponding peptide having a free amino group and protected carboxy groups in a solvent as used in peptide chemistry and with the addition of 3-hydroxy-4-oxo-3,4-di-hydro-1,2,3-benzotriazine (HOObt) and a carbodiimide and then splitting off the Dnp-(2,4-dinitrophenyl) group. The invention also provides a new dipeptide, i.e. ?Glu-His(Dnp)-OH.
USING ?Glu-His(DnP)-OH
Abstract of the disclosure:
Process for the manufacture of LH-RH andLH-RH ana-logs, which comprises reacting ?Glu-His(DnP)-OH with the corresponding peptide having a free amino group and protected carboxy groups in a solvent as used in peptide chemistry and with the addition of 3-hydroxy-4-oxo-3,4-di-hydro-1,2,3-benzotriazine (HOObt) and a carbodiimide and then splitting off the Dnp-(2,4-dinitrophenyl) group. The invention also provides a new dipeptide, i.e. ?Glu-His(Dnp)-OH.
Description
It is the object of the present invention to provide a process for the manufacture of LH-RH and LH-RH analogs, which comprises reacting rGlu-His(Dnp)-OH with the cor-responding peptide having a free amino group ana protected carboxy groups in a solvent as used in peptide chemistry and with the addition of 3 -hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt) and a carbodiimide and then splitting off the Dnp-(2,4-dinitrophenyl) group.
It is known that LH-RH is a hormone of the hypothala-mus of the formula I
rGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, (I) releasing the gonadotropic hormones LH and FSH in the pi-tuitary gland. LH-RH analogs are peptides in which indivi-dual or several amino acids of LH-RH are replaced and/or the peptide chain is modified by shortening, lengthening and/or derivatization. The substitution of glycine in position 6 by D-amino acids and in position 10 by alkyl amines has gained special importance.
Suitable second components in theprocess of the in-vention are, therefore, mainly octa- and hepta-peptides of the formulae II: H-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-NH2 III: H-Trp-Ser-Tyr-X-Leu-Arg-Pro-NH-C2H5 in which X denotes Gly or D-amino acids or derivatives thereof, for example D-Ser(But), D-Leu, D-Ala, D-Phe, D-Trp, D-Gln(cyclohexyl), D-Glu(OBu ) and D-Lys(Boc).
The novel dipeptide derivative rGlu-His-(Dnp)-OH used as starting component is prepared in usual manner, for ex-ample by reacting rGlu-His-OH with 2,4-dinitrofluoroben-zene in aqueous solution buffered with NaHCO3.
For reasons of solubility polar solvents, for example dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide, - phosphoric acid tris(dimethyl amide) or N-methyl-pyrroli-done, are suitable as solvents in the process of the inven-tion.
~1~9378 The synthesis can be carried out at a temperature in the range of from -10 C and room temperature. The reaction is preferably started at about 0C and the temperature is then allowed to rise to room temperature.
As condensing agent dicyclohexyl carbodiimide (DCC), which is readily accessible, is preferred, although other carbodiimides, for example l~cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide toluene sulfonate or l-ethyl-3-(3-di-methylaminopropyl)-carbodiimide hydrochloride can also be used.
As intermediates peptides protected by the dinitro-phenyl radical at the imidazole ring o'f the histidine are first obtained. This dinitrophenyl radical can be split off by methods known in peptide chemistry, for example by thiolysis (Biochem.-Biophys. Res.Commun. 29, 178 (1967);
Biochemistry 9, 5122 (1970)) or by hydrazinolysis (Tetra-hydron Letters 44, 4121 (1971)).
In general, the Dnp-containing intermediate need not be isolated. After the peptide coupling a thiol, for ex-ample mercaptoethanol or ethylmercaptan, or hydrazine, is added to the reaction mixture and after splitting off of the Dnp radical, which can be readily ascertained by thin layer chromatogram, the mixture is worked up. If hydrazine is used as deblocking reagent, dimethyl formamide is unsui table as solvent for it is not stable with respect to hy-drazine.
It is known that LH-RH and the analogs thereof can be produced especially be combining rGlu-His-OH with ap-propriate peptides by mean of DCC with the addition of N-hydroxy-compounds reducing racemization, for example N-hydroxysuccinimide (HONSu) l-hydroxybenzotriazole (HOBt), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt), or N-hydroxy-5-norbornene-endo-2,3-dicarboximide (HNOB) (cf. Biochem-Biophys.Res.Commun 45, 767 - 773 35 (1971)).
High pressure liquid chromatography (HPLC) has reveal-ed, however, that in this process a partial racemization .. . . . . . .. . . . . . . . .. .. . .
'L~'~9378 of the histidine cannot be avoided (cf. Tables 1 and 2).
For reducing the racemization of the histidine in the syn-thesis of histidine peptides the protection of the imida-zole ring by the tosyl group (Tos group) is reco~mended in literature. A corresponding protection by the 2,4-dinitro-phenyl group (Dnp-group) appears to be unsuitable because of too important a racemization of the histidines and the tendency to further contamination (cf. Rec.Trav.Chim.Pay-Bas 93, 256 (1974)).
It has, therefore, been surprising that the racemiza-tion of the histidine can be reduced to less than 2 ~ of D-histidine when the histidine in the starting compound rGlu-His-OH is protected by the Nim-2,4-dinitrophenyl group (Dnp) and the reaction is carried out under the con-ditions of the invention. Under the same conditions theracemization could not be reduced below 5 ~ of D-histidine using the tosyl group for N protection.
Comparative tests in the LH-RH synthesis using diffe-rent carboxy components and various condensation methods are indicated in Table 1. In the tests the content of [D-His ]-LH-RH is determined by high pressure liquid chro-matography (HPLC). The table shows that only the use of DCC-HOObt and t~e N -Dnp-protection make it possible to repress the racemization to a value below 2 % of D-hista-mine.
The same results are obtained in the synthesis of aLH-RH analog as can be seen from Table 2.
Table 1 Study of racemization in the LH-RH synthesis The reaction conditions are specified in Examples 3 to 5. HPLC: column (0.4 x 25 cm filled with LiChrosorb( ) SI 60 (silica gel of an average pore width of 60 ~) of Messrs. Merck, Federal Republic of Germany; eluent 310 parts by volume of chloroform, 190 parts by volume of methanol, 14 parts by volume of water, 3.1 parts by volume of triethylamine and 1 part by volume of formic acid:
elution rate 1 ml/minute;
RT (LH-RH) = about 14.0 to 14.5 minutes RT ([D his ]LH-RH) = about 17.5 to 18 minutes carboxy component method % [D-His 3LH-RH
r Glu-His-OH DCC/HOBt 23 " DCC/HONB 15 DCC/HONSu 15 " DCC/HOObt 12 r Glu-Hls(Tos)-OH DCC/HONSu 9 " DCC/HOObt 5 ~Glu-His(Dnp)-OH DCC/HOBt - 25 " DCC/HONB 7 " DCC/HONSu 6 " DCC/HOObt 1-2 Table 2 Study of racemization in the synthesis of [D-Ser(Bu ) ]LH-RH-(l-9) nonapeptide ethylamide The reaction conditions are specified in Examples 6 and 7. HPLC: column (0.4 x 25 cm) filled with LiChro-sorb( )SI 60 of Messrs. Merck; eluent: 410 parts by volume of acetonitrile, 29 parts by weight of methanol, 29 parts by volume of water, 20 parts by volume of chloroform, 3.7 parts by volume of triethylamine, 1 part by volume of formic acid; elution rate: 2 ml/minute; R ([D-Ser(Bu ) ]-LH-RH-(l-9)-nonapeptide ethyl amide) ~ 40 minutes, RT ([D-His , D-Ser(Bu ) ]LH-RH-(l,9)-nonapeptide ethyl amide ) = 48 minutes carboxy component method % D-His2 compound r Glu-His-OH DCC/HOBt 20 " DCC/HONSu 11 " DCC/HONB 11 " DCC/HOObt 6 X
.~L1~9378 arboxy componentmethod % D-His2 compound Glu-His(Dnp)-OH DCC/HOBt 12 " DCC/HONSu 4 " DCC/HONB 3 " DCC/HOObt l-1.5 The following examples illustrate the invention.
E X A M P L E 1:
Preparation of rGlu-His(Dnp)-OH 0.5 H2O
30 g (100 mmols) of rGlu-His-O~ H2O and 20 g (238 mmols) of NaHC03 are dissolved in 200 ml of water and, while stirring, a solution of 22.3 g (120 mmols) of 2,4-dinitro-fluorobenzene in 100 ml of dioxane is added dropwise over a period of l hour. Stirring of the mixture is continued for 3 hours at room temperature. The precipitate formed is fil-tered off with suction and the filtrate is extracted twice, each time with 200 ml of ethyl acetate. The aqueous phase is concentrated, the residue is dissolved in 200 ml of water and 120 ml of 1 N HCl are added. The upper layer is sepa-rated by decantation from the precipitating red oil and filtered. 300 ml of n-butanol are added to the aqueous solution, the mixture is thoroughly shaken and the phase mixture is left to stand overnight at 4C. On the follow-ing day the yellow precipitate ls filtered off with suction and washed with a small amount of n-butanol. For a quanti-tative removal of Glu-His-OH still present the preci-pitate is stirred twice at room temperature, each time for one hour with 300 ml of water, the mixture is filtered with suction and the residue is dried over P2O5 under reduced pressure. Yield: 20 g; melting point 235 to 241C with decomposition.
[~] 1 - -12.2C (c = 1, in acetic acid) [CL]~l - +18.9C (c = l, in dimethyl formamide) X
E X A M P L E 2:
Preparation of ~lu-His(Tos)-OH as comparative substance for Table 1 3 g (10 mmols) of rGlu-His-OH and 2.3 g of NaHCO3 are dissolved in 20 ml of water and, while stirring at room temperature, a solution of 2.1 g (10 % excess) of p-toluene sulfochloride in about 10 ml of dioxane is slowly added drop by drop. When the addition is terminated, stirring of the mixture is continued for a further hour, whereupon it is extracted twice with ether. The aqueous phase is acidified to pH 2 with 2N HCl and the precipitate is fil-tered off with suction. It is washed with water and the filter residue is dried over P2O5 under reduced pressure.
Yield: 2.2 g.
E X A M P L E 3:
Synthesis of LH-RH with rGlu-His-OH using the different coupling methods as indicated in Table 1 640 mg of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 di-tosylate and 150 ml of Glu-His-OH H2O are dissolved in 3 ml ofdimethyl acetamide. 0.065 ml ofN-ethylmorpholine and 68 mg of HOBt (or 81 mg of HOObt or 90 mg of HONB or 57 mg Gf HONSu) are added to the solution. The mixture is cooled to 0C, 110 mg of DCC are added, the whole is stir-red for 1 hour at 0C and then overnight at room tempera-ture. On the following day 0.1 ml of hydrazine hydrate is added, the mixture is stirred for 2 hours at room tempera-ture and then filtered off with suction. The filtrate is partitioned between 30 ml of n-butanol and 30 ml of NaHCO3 solution. The n-butanol phase is concentrated in a high vacuum and the residue is triturated with ether. The pre-cipitate is filtered off with suction and dried. Next, the substance is dissolved in dilute acetic acid and the solution is chromatographed over approximately 10 ml Dowex 1 x 2 (acetate form). The column is eluted with water and the eluate is lyophilized. Yield 500 - 600 mg.
For further purification the crude LH-RH is subjected to a partition chromatography on a hydroxypropylated cross-'~,i, ~1~937~
linked dextran gel as described in Example le of German Auslegeschrift 2,438,350. Yield of pure LH-RH: about 250 - 300 mg.
E X A M P L E 4:
Synthesis of LH-RH with ~Glu-His(Tos)-OH using the different coupling_methods as indicated in Table 1 640 mg of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 di-tosylate and 210 mg of rGlu-His(Tos)-OH are dissolved in 3 ml of dimethyl acetamide~ 0.065 ml of N-ethylmorpholine and 81 mg of HOObt (or 57 mg of HONSu) are added whereupon the mixture is cooled to 0C. 110 mg of DCC are then added and the mixture is stirred for 1 hour at 0C and overnight at room temperature. The mixture is further worked up and purified as described in Example 3. Yield: 275 mg (or 250 mg).
E X A M P L E 5:
Synthesis of LH-RH with rGlu-His(Dnp)-OH using different coupling methods according to Table 1 11 g (25 mmols) of rGlu-His(Dnp)-OH 0.5 H2O and 32 g (25 mmols) of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 2 TosOH are dissolved in 150 ml of dimethyl acetamide. 4.07 g of HOObt (3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotria-zine) (or 3.4 g of HOBt or 4.5 g H~NB or 2.9 g of H~NSu) are added, the mixture is cooled to 0C, whereupon 3.25 ml of N-ethylmorpholine and 5.5 g of DCC are added. The mix-ture is stirred for 1 hour at 0C and then overnight at room temperature. The precipitate formed is filtered off with suction and washed with a small amount of dimethyl acetamide. 2.5 mlof hydrazine hydrate of '00 % strength are added to the filtrate and the whole is stirred for 4 hours at room temperature. A black solution is formed.
The peptide is precipitated with 1,250 ml of ethyl acetate.
The precipitate is filtered off with suction and washed with ethyl acetate. It is then dissolved in 430 ml of methanol and the peptide is precipitated again with 1,400 ml of ethyl acetate. The precipitate is filtered off with suction, dissolved in 1,000 ml of n-butanol and shaken ~1 .
3'7~3 once with l,000 ml of saturated NaHCO3 solution and then with 750 ml of saturated NaHCO3 solution. The n-butanol phase is concentrated in a high vacuum. The residue is triturated with ethyl acetate and dried. Crude yield:
about 25 g. The substance is further purified as describ-ed in Example 3. Yield of pure substance: 12 to 14 g.
E X A ~ P L E 6:
Synthesis of [D-Ser(But)6]LH-RH-(l-9)-nonapeptide ethyl amide with ~Glu-His(Dnp)-OH using different coupling ~ethods as indicated in Table 2 11 g (25 mmols) of rGlu-His(Dnp)OH 0.5 H2O and 26.6 g (25 mmols) of H-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5 2 HCl are dissolved in 150 ml of dimethyl acet-amide. 4.07 g of 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzo-triazine (HOObt) (or 3.4 g of HOBt or 4.5 g of HONB or 2.9 g of HONSu) are added and the mixture is cooled to 0C.
Next, 3.25 ml of N-ethylmorpholine and 5.5 g of DCC are added and the mixture is stirred for 1 hour at OC and overnight at room temperature. The precipitate formed is filtered off with suction and washed with 50 ml of dime-thyl acetamide. 2.5 ml of hydrazine hydrate of 100 ~
strength are added to the filtrate, whereupon the mixture is stirred for 4 hours at room temperature and then the peptide is precipitate with 1,250 ml of ethyl acetate.
me precipitate is filtered off with suction and thoroughly washed with ethyl acetate. Next, the substance is dissolv-ed in 430 ml of methanol. The peptide is precipitated again by adding 1,400 ml of ethyl acetate. The precipitate is dissolved in l,000 ml of n-butanol and the solution is ex-tracted once with l,000 ml of saturated NaHCO3 solution andthen with 750 ml of saturated NaHCO3 solution. The n-buta-nol phase is concentrated and the residue is triturated with ethyl acetate. The precipitate is filtered off with suction, washed with ethyl acetate and dried. Yield:
18.7 g~
For conversion into the acetate the above substance is dissolved in 50 ml of water and 5 ml of acetic acid and 93~3 chromatographed over 230 ml of Dowex lx2 (acetate form).
The column is eluted with water. The fractions contain-ing the substance are combined and lyophilized . Yield:
about 15.0 g.
[~]D3= -40C (c~ 1, in methanol) The substance is further purified as described in Ex-ample 3.
Yield: about 9.5 to 11 g E X A M P L E 7:
Synthesis of [D-Ser-(But) ]LH-RH-(l-9)-nonapeptide ethyl amide with ~Glu-His-OH using different coupling methods as indicated in Table 2 .
532 mg of H-Trp-Ser-Tyr-D-Ser-(But)-Leu-Arg-Pro-NHC2H5 2 HCl and 150 mg of rGlu-His-OH H2O are dis-lS solved in 3 ml of dimethyl acetamide. 0.065 ml of N-ethyl-morpholine and 68 mg of HOBt (or 81 mb of HOOBt or 90 mg of HONB or 57 mg of HONSu) are added. The mixture is cool-ed to 0C, 110 mg of DCC are added and the whole is stir-red first for 1 hour at 0C and then overnight at room tem-perature.
The substance is further worked up and purified as described in Example 3. Yield: 104 to 207 mg.
This application is a division of Canadian patent application number 345,595, filed February 13, 1980.
It is known that LH-RH is a hormone of the hypothala-mus of the formula I
rGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, (I) releasing the gonadotropic hormones LH and FSH in the pi-tuitary gland. LH-RH analogs are peptides in which indivi-dual or several amino acids of LH-RH are replaced and/or the peptide chain is modified by shortening, lengthening and/or derivatization. The substitution of glycine in position 6 by D-amino acids and in position 10 by alkyl amines has gained special importance.
Suitable second components in theprocess of the in-vention are, therefore, mainly octa- and hepta-peptides of the formulae II: H-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-NH2 III: H-Trp-Ser-Tyr-X-Leu-Arg-Pro-NH-C2H5 in which X denotes Gly or D-amino acids or derivatives thereof, for example D-Ser(But), D-Leu, D-Ala, D-Phe, D-Trp, D-Gln(cyclohexyl), D-Glu(OBu ) and D-Lys(Boc).
The novel dipeptide derivative rGlu-His-(Dnp)-OH used as starting component is prepared in usual manner, for ex-ample by reacting rGlu-His-OH with 2,4-dinitrofluoroben-zene in aqueous solution buffered with NaHCO3.
For reasons of solubility polar solvents, for example dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide, - phosphoric acid tris(dimethyl amide) or N-methyl-pyrroli-done, are suitable as solvents in the process of the inven-tion.
~1~9378 The synthesis can be carried out at a temperature in the range of from -10 C and room temperature. The reaction is preferably started at about 0C and the temperature is then allowed to rise to room temperature.
As condensing agent dicyclohexyl carbodiimide (DCC), which is readily accessible, is preferred, although other carbodiimides, for example l~cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide toluene sulfonate or l-ethyl-3-(3-di-methylaminopropyl)-carbodiimide hydrochloride can also be used.
As intermediates peptides protected by the dinitro-phenyl radical at the imidazole ring o'f the histidine are first obtained. This dinitrophenyl radical can be split off by methods known in peptide chemistry, for example by thiolysis (Biochem.-Biophys. Res.Commun. 29, 178 (1967);
Biochemistry 9, 5122 (1970)) or by hydrazinolysis (Tetra-hydron Letters 44, 4121 (1971)).
In general, the Dnp-containing intermediate need not be isolated. After the peptide coupling a thiol, for ex-ample mercaptoethanol or ethylmercaptan, or hydrazine, is added to the reaction mixture and after splitting off of the Dnp radical, which can be readily ascertained by thin layer chromatogram, the mixture is worked up. If hydrazine is used as deblocking reagent, dimethyl formamide is unsui table as solvent for it is not stable with respect to hy-drazine.
It is known that LH-RH and the analogs thereof can be produced especially be combining rGlu-His-OH with ap-propriate peptides by mean of DCC with the addition of N-hydroxy-compounds reducing racemization, for example N-hydroxysuccinimide (HONSu) l-hydroxybenzotriazole (HOBt), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt), or N-hydroxy-5-norbornene-endo-2,3-dicarboximide (HNOB) (cf. Biochem-Biophys.Res.Commun 45, 767 - 773 35 (1971)).
High pressure liquid chromatography (HPLC) has reveal-ed, however, that in this process a partial racemization .. . . . . . .. . . . . . . . .. .. . .
'L~'~9378 of the histidine cannot be avoided (cf. Tables 1 and 2).
For reducing the racemization of the histidine in the syn-thesis of histidine peptides the protection of the imida-zole ring by the tosyl group (Tos group) is reco~mended in literature. A corresponding protection by the 2,4-dinitro-phenyl group (Dnp-group) appears to be unsuitable because of too important a racemization of the histidines and the tendency to further contamination (cf. Rec.Trav.Chim.Pay-Bas 93, 256 (1974)).
It has, therefore, been surprising that the racemiza-tion of the histidine can be reduced to less than 2 ~ of D-histidine when the histidine in the starting compound rGlu-His-OH is protected by the Nim-2,4-dinitrophenyl group (Dnp) and the reaction is carried out under the con-ditions of the invention. Under the same conditions theracemization could not be reduced below 5 ~ of D-histidine using the tosyl group for N protection.
Comparative tests in the LH-RH synthesis using diffe-rent carboxy components and various condensation methods are indicated in Table 1. In the tests the content of [D-His ]-LH-RH is determined by high pressure liquid chro-matography (HPLC). The table shows that only the use of DCC-HOObt and t~e N -Dnp-protection make it possible to repress the racemization to a value below 2 % of D-hista-mine.
The same results are obtained in the synthesis of aLH-RH analog as can be seen from Table 2.
Table 1 Study of racemization in the LH-RH synthesis The reaction conditions are specified in Examples 3 to 5. HPLC: column (0.4 x 25 cm filled with LiChrosorb( ) SI 60 (silica gel of an average pore width of 60 ~) of Messrs. Merck, Federal Republic of Germany; eluent 310 parts by volume of chloroform, 190 parts by volume of methanol, 14 parts by volume of water, 3.1 parts by volume of triethylamine and 1 part by volume of formic acid:
elution rate 1 ml/minute;
RT (LH-RH) = about 14.0 to 14.5 minutes RT ([D his ]LH-RH) = about 17.5 to 18 minutes carboxy component method % [D-His 3LH-RH
r Glu-His-OH DCC/HOBt 23 " DCC/HONB 15 DCC/HONSu 15 " DCC/HOObt 12 r Glu-Hls(Tos)-OH DCC/HONSu 9 " DCC/HOObt 5 ~Glu-His(Dnp)-OH DCC/HOBt - 25 " DCC/HONB 7 " DCC/HONSu 6 " DCC/HOObt 1-2 Table 2 Study of racemization in the synthesis of [D-Ser(Bu ) ]LH-RH-(l-9) nonapeptide ethylamide The reaction conditions are specified in Examples 6 and 7. HPLC: column (0.4 x 25 cm) filled with LiChro-sorb( )SI 60 of Messrs. Merck; eluent: 410 parts by volume of acetonitrile, 29 parts by weight of methanol, 29 parts by volume of water, 20 parts by volume of chloroform, 3.7 parts by volume of triethylamine, 1 part by volume of formic acid; elution rate: 2 ml/minute; R ([D-Ser(Bu ) ]-LH-RH-(l-9)-nonapeptide ethyl amide) ~ 40 minutes, RT ([D-His , D-Ser(Bu ) ]LH-RH-(l,9)-nonapeptide ethyl amide ) = 48 minutes carboxy component method % D-His2 compound r Glu-His-OH DCC/HOBt 20 " DCC/HONSu 11 " DCC/HONB 11 " DCC/HOObt 6 X
.~L1~9378 arboxy componentmethod % D-His2 compound Glu-His(Dnp)-OH DCC/HOBt 12 " DCC/HONSu 4 " DCC/HONB 3 " DCC/HOObt l-1.5 The following examples illustrate the invention.
E X A M P L E 1:
Preparation of rGlu-His(Dnp)-OH 0.5 H2O
30 g (100 mmols) of rGlu-His-O~ H2O and 20 g (238 mmols) of NaHC03 are dissolved in 200 ml of water and, while stirring, a solution of 22.3 g (120 mmols) of 2,4-dinitro-fluorobenzene in 100 ml of dioxane is added dropwise over a period of l hour. Stirring of the mixture is continued for 3 hours at room temperature. The precipitate formed is fil-tered off with suction and the filtrate is extracted twice, each time with 200 ml of ethyl acetate. The aqueous phase is concentrated, the residue is dissolved in 200 ml of water and 120 ml of 1 N HCl are added. The upper layer is sepa-rated by decantation from the precipitating red oil and filtered. 300 ml of n-butanol are added to the aqueous solution, the mixture is thoroughly shaken and the phase mixture is left to stand overnight at 4C. On the follow-ing day the yellow precipitate ls filtered off with suction and washed with a small amount of n-butanol. For a quanti-tative removal of Glu-His-OH still present the preci-pitate is stirred twice at room temperature, each time for one hour with 300 ml of water, the mixture is filtered with suction and the residue is dried over P2O5 under reduced pressure. Yield: 20 g; melting point 235 to 241C with decomposition.
[~] 1 - -12.2C (c = 1, in acetic acid) [CL]~l - +18.9C (c = l, in dimethyl formamide) X
E X A M P L E 2:
Preparation of ~lu-His(Tos)-OH as comparative substance for Table 1 3 g (10 mmols) of rGlu-His-OH and 2.3 g of NaHCO3 are dissolved in 20 ml of water and, while stirring at room temperature, a solution of 2.1 g (10 % excess) of p-toluene sulfochloride in about 10 ml of dioxane is slowly added drop by drop. When the addition is terminated, stirring of the mixture is continued for a further hour, whereupon it is extracted twice with ether. The aqueous phase is acidified to pH 2 with 2N HCl and the precipitate is fil-tered off with suction. It is washed with water and the filter residue is dried over P2O5 under reduced pressure.
Yield: 2.2 g.
E X A M P L E 3:
Synthesis of LH-RH with rGlu-His-OH using the different coupling methods as indicated in Table 1 640 mg of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 di-tosylate and 150 ml of Glu-His-OH H2O are dissolved in 3 ml ofdimethyl acetamide. 0.065 ml ofN-ethylmorpholine and 68 mg of HOBt (or 81 mg of HOObt or 90 mg of HONB or 57 mg Gf HONSu) are added to the solution. The mixture is cooled to 0C, 110 mg of DCC are added, the whole is stir-red for 1 hour at 0C and then overnight at room tempera-ture. On the following day 0.1 ml of hydrazine hydrate is added, the mixture is stirred for 2 hours at room tempera-ture and then filtered off with suction. The filtrate is partitioned between 30 ml of n-butanol and 30 ml of NaHCO3 solution. The n-butanol phase is concentrated in a high vacuum and the residue is triturated with ether. The pre-cipitate is filtered off with suction and dried. Next, the substance is dissolved in dilute acetic acid and the solution is chromatographed over approximately 10 ml Dowex 1 x 2 (acetate form). The column is eluted with water and the eluate is lyophilized. Yield 500 - 600 mg.
For further purification the crude LH-RH is subjected to a partition chromatography on a hydroxypropylated cross-'~,i, ~1~937~
linked dextran gel as described in Example le of German Auslegeschrift 2,438,350. Yield of pure LH-RH: about 250 - 300 mg.
E X A M P L E 4:
Synthesis of LH-RH with ~Glu-His(Tos)-OH using the different coupling_methods as indicated in Table 1 640 mg of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 di-tosylate and 210 mg of rGlu-His(Tos)-OH are dissolved in 3 ml of dimethyl acetamide~ 0.065 ml of N-ethylmorpholine and 81 mg of HOObt (or 57 mg of HONSu) are added whereupon the mixture is cooled to 0C. 110 mg of DCC are then added and the mixture is stirred for 1 hour at 0C and overnight at room temperature. The mixture is further worked up and purified as described in Example 3. Yield: 275 mg (or 250 mg).
E X A M P L E 5:
Synthesis of LH-RH with rGlu-His(Dnp)-OH using different coupling methods according to Table 1 11 g (25 mmols) of rGlu-His(Dnp)-OH 0.5 H2O and 32 g (25 mmols) of H-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 2 TosOH are dissolved in 150 ml of dimethyl acetamide. 4.07 g of HOObt (3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotria-zine) (or 3.4 g of HOBt or 4.5 g H~NB or 2.9 g of H~NSu) are added, the mixture is cooled to 0C, whereupon 3.25 ml of N-ethylmorpholine and 5.5 g of DCC are added. The mix-ture is stirred for 1 hour at 0C and then overnight at room temperature. The precipitate formed is filtered off with suction and washed with a small amount of dimethyl acetamide. 2.5 mlof hydrazine hydrate of '00 % strength are added to the filtrate and the whole is stirred for 4 hours at room temperature. A black solution is formed.
The peptide is precipitated with 1,250 ml of ethyl acetate.
The precipitate is filtered off with suction and washed with ethyl acetate. It is then dissolved in 430 ml of methanol and the peptide is precipitated again with 1,400 ml of ethyl acetate. The precipitate is filtered off with suction, dissolved in 1,000 ml of n-butanol and shaken ~1 .
3'7~3 once with l,000 ml of saturated NaHCO3 solution and then with 750 ml of saturated NaHCO3 solution. The n-butanol phase is concentrated in a high vacuum. The residue is triturated with ethyl acetate and dried. Crude yield:
about 25 g. The substance is further purified as describ-ed in Example 3. Yield of pure substance: 12 to 14 g.
E X A ~ P L E 6:
Synthesis of [D-Ser(But)6]LH-RH-(l-9)-nonapeptide ethyl amide with ~Glu-His(Dnp)-OH using different coupling ~ethods as indicated in Table 2 11 g (25 mmols) of rGlu-His(Dnp)OH 0.5 H2O and 26.6 g (25 mmols) of H-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5 2 HCl are dissolved in 150 ml of dimethyl acet-amide. 4.07 g of 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzo-triazine (HOObt) (or 3.4 g of HOBt or 4.5 g of HONB or 2.9 g of HONSu) are added and the mixture is cooled to 0C.
Next, 3.25 ml of N-ethylmorpholine and 5.5 g of DCC are added and the mixture is stirred for 1 hour at OC and overnight at room temperature. The precipitate formed is filtered off with suction and washed with 50 ml of dime-thyl acetamide. 2.5 ml of hydrazine hydrate of 100 ~
strength are added to the filtrate, whereupon the mixture is stirred for 4 hours at room temperature and then the peptide is precipitate with 1,250 ml of ethyl acetate.
me precipitate is filtered off with suction and thoroughly washed with ethyl acetate. Next, the substance is dissolv-ed in 430 ml of methanol. The peptide is precipitated again by adding 1,400 ml of ethyl acetate. The precipitate is dissolved in l,000 ml of n-butanol and the solution is ex-tracted once with l,000 ml of saturated NaHCO3 solution andthen with 750 ml of saturated NaHCO3 solution. The n-buta-nol phase is concentrated and the residue is triturated with ethyl acetate. The precipitate is filtered off with suction, washed with ethyl acetate and dried. Yield:
18.7 g~
For conversion into the acetate the above substance is dissolved in 50 ml of water and 5 ml of acetic acid and 93~3 chromatographed over 230 ml of Dowex lx2 (acetate form).
The column is eluted with water. The fractions contain-ing the substance are combined and lyophilized . Yield:
about 15.0 g.
[~]D3= -40C (c~ 1, in methanol) The substance is further purified as described in Ex-ample 3.
Yield: about 9.5 to 11 g E X A M P L E 7:
Synthesis of [D-Ser-(But) ]LH-RH-(l-9)-nonapeptide ethyl amide with ~Glu-His-OH using different coupling methods as indicated in Table 2 .
532 mg of H-Trp-Ser-Tyr-D-Ser-(But)-Leu-Arg-Pro-NHC2H5 2 HCl and 150 mg of rGlu-His-OH H2O are dis-lS solved in 3 ml of dimethyl acetamide. 0.065 ml of N-ethyl-morpholine and 68 mg of HOBt (or 81 mb of HOOBt or 90 mg of HONB or 57 mg of HONSu) are added. The mixture is cool-ed to 0C, 110 mg of DCC are added and the whole is stir-red first for 1 hour at 0C and then overnight at room tem-perature.
The substance is further worked up and purified as described in Example 3. Yield: 104 to 207 mg.
This application is a division of Canadian patent application number 345,595, filed February 13, 1980.
Claims (3)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of ?Glu-His(DNP)-OH in which ?Glu-His-OH is reacted with 2,4-dinitrofluorobenzene in an aqueous buffered solution.
2. A process as claimed in claim 1 in which the aqueous solution is buffered with sodium bicarbonate.
3. The dipeptide ?Glu-His(DNP)-OH whenever obtained accord-ing to a process as claimed in claim 1 or claim 2 or by an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000406187A CA1149378A (en) | 1979-02-14 | 1982-06-28 | Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2905502A DE2905502C2 (en) | 1979-02-14 | 1979-02-14 | Process for the production of LH-RH or LH-RH analogues and pyroglutamyl-N → i → m → -dinitrophenyl-histidine |
| DEP2905502.5 | 1979-02-14 | ||
| CA000345595A CA1144160A (en) | 1979-02-14 | 1980-02-13 | Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh |
| CA000406187A CA1149378A (en) | 1979-02-14 | 1982-06-28 | Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1149378A true CA1149378A (en) | 1983-07-05 |
Family
ID=27166584
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000406187A Expired CA1149378A (en) | 1979-02-14 | 1982-06-28 | Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1149378A (en) |
-
1982
- 1982-06-28 CA CA000406187A patent/CA1149378A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4100274A (en) | Polypeptide | |
| US3992365A (en) | Agonist analogues of luteinizing hormone releasing hormone | |
| US4275001A (en) | Process for the manufacture of LH-RH and LH-RH analogs using Glu-His(DnP)-OH | |
| US4086219A (en) | Nonapeptides and methods for their production | |
| US3817973A (en) | Process for the preparation of cyclopeptides derived from polymyxins | |
| US3917578A (en) | Process for producing somatostatin and intermediates | |
| CA1137467A (en) | Process for the preparation of somatostatin | |
| US4124703A (en) | Luliberin analogs | |
| CA1149378A (en) | Process for the manufacture of lh-rh and lh-rh analogs using glu-his(dnp)-oh | |
| US3971736A (en) | Cathepsin in D inhibitors | |
| US3826794A (en) | Protected decapeptide derivatives of gonadotropin releasing hormone | |
| US3780014A (en) | Octapeptide intermediate to gonadotropin releasing hormone | |
| FUJII et al. | Studies on Peptides. LII. Application of the Trifluoromethanesulphonic Acid Procedure to the Synthesis of a Peptide with Somatostatin Activity | |
| US3388112A (en) | Acth active peptides modified at the nu-terminal position | |
| Kobayashi et al. | Oxytocin Analogues Containing No Disulfide Bond. I. Synthesis of the Lactam of L-Tyrosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-L-α-aminosuberyl-L-prolyl-L-leucylglycine Amide | |
| Hofmann et al. | Studies with the S-peptide-S-protein system: The role of glutamic acid-2, lysine-7, and methionine-13 in S-peptide1–14 for binding to and activation of S-protein | |
| Bodanszky et al. | Cholecystokinin-pancreozymin. I. Synthesis of peptides corresponding to the N-terminal sequence | |
| US4043993A (en) | New pentapeptides and methods for their production | |
| Inouye | Syntheses of Peptides Related to the N-Terminal Structure of Corticotropin. V. Synthesis of the Peptide Fragments, His-Phe-Arg-Try, His-Phe-Arg-Try-Gly and Glu-His-Phe-Arg-Try-Gly | |
| US4128540A (en) | Pyroglutamyl-histidyl-tryptophanyl-seryl-tyrosyl hydrazides | |
| US3778427A (en) | Hexapeptide | |
| US3781272A (en) | Tripeptide | |
| Ferderigos et al. | The effect of modifications of the A 5 and A 19 amino acid residues on the biological activity of insulin.[Leu 5-A] and [Phe 19-A] sheep insulins | |
| KR850000411B1 (en) | Process for the preparation of lh-rh or lh-rh analogs | |
| US3752799A (en) | Nalpha-acetyl-2-o-methyltyrosine-oxytocin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |