CA1074300A - Method for removal of thiol-protecting groups - Google Patents
Method for removal of thiol-protecting groupsInfo
- Publication number
- CA1074300A CA1074300A CA279,924A CA279924A CA1074300A CA 1074300 A CA1074300 A CA 1074300A CA 279924 A CA279924 A CA 279924A CA 1074300 A CA1074300 A CA 1074300A
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- acid
- cys
- phe
- dimethylformamide
- boc
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
- C07K1/067—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for sulfur-containing functions
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/655—Somatostatins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Endocrinology (AREA)
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- Gastroenterology & Hepatology (AREA)
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- Peptides Or Proteins (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A novel method for removal of protective group(s) from an amino acid or peptide having thiol group(s) protected with p-methoxybenzyl, 1-adamantyl or t-butyl group by treating the protected amino acid or peptide with mercuric salt of acetic acid or halogenoacetic acid, the removal being effected selectively and very smoothly in a good yield under extremely mild condition without giving any undesirable influence upon other functional or protective group(s).
A novel method for removal of protective group(s) from an amino acid or peptide having thiol group(s) protected with p-methoxybenzyl, 1-adamantyl or t-butyl group by treating the protected amino acid or peptide with mercuric salt of acetic acid or halogenoacetic acid, the removal being effected selectively and very smoothly in a good yield under extremely mild condition without giving any undesirable influence upon other functional or protective group(s).
Description
10';~ 30V
Method ~or removal of thlol-protecting groups SPECIFICATION
This invention relates to a novel and useful method for removal of thiol-protecting groups.
More specifically, the present invention is directed to a method for removal o~ protective group(s) from an amino acid or peptide havlng thiol group(s) protected with p-methoxybenzyl, l-adamantyl or t-butyl.
Generally, in organic reactions it is often necessary to protect and, then, regenerate a thiol group. Especially in peptide synthesis, protection and regeneration of thiol groups are quite important.
Among procedures of protecting a thiol group and, then, removing the protective group, most widely available procedures are in these days the procedure of protectlng lt with a benzyl group and, then, removing this protective group wlth metallic sodium in liquid ammonia and of protecting the thiol with p-methoxybenzyl and then removing the latter with refluxing tri-fluoroacetic acid or anhydrous hydrogen fluoride ["J. Biol. Chem. Vol. 108, 753-761 (1935)", Bull. Chem.
Soc. Japan, Vol. 37, No. 39 433-434 (1964)", and "Bull.
Chem. Soc. Japan, Vol. 40, 2164-2167 ~1967)"~.
However, because the condit~ons employed in the removal .; -.. . ~
.
107~300 of such protective groups are somewhat drastic, there are cases in which it is found to be impossible to selectively remove the thiol-protecting group with the other functional groups and protective groups being kept intact.
Because t-butyl and l-adamantyl groups may be easily introduced to the thiol group, attempts have been made to utilize them as protective groups for thiol, only to find, however, that these protective groups are very difficult to remove subsequently ["J. A. C. S. Vol.
8~, 201-207 (1963)"]. Thus, no effective procedure for removing such protective groups has been known.
Under these circumstances we sought for a new method of regenerating a thiol group protected by p-methoxybenzyl, l-adamantyl or t-butyl and have discovered that such protective groups may be removed with extreme ease and in good yield by treating the substrate under very mild con~itions, i.e. with a mercuric salt of a carboxylic acid of the formula CX3COOH (I) wherein X is hydrogen, chlorine or fluorine.
Developed as a sequel of the above finding, this invention relates to a method for removal of a protective group from an amino acid or peptide having thiol group(s) protected with p methoxybenzyl, l-adamantyl or t-butyl characterized in that the protected amino acid or peptide is treated with a mercuric salt of a ~, .
1~7430~
carboxylic acid (I).
In accordance with the invention~ an amino acid or peptide having a thiol group or thiol groups protected with p-methoxybenzyl, l-adamantyl or t-butyl is treated with a mercuric salt of a carboxylic acid (I).
The present method is applicable to any amino acids having thiol group(s) protected with the protective group(s) and those amino acids may be exemplified by cysteine or homocysteine. The present method ls also applicable to any peptides having thiol group(s) protected with the protective group(s).
These peptides may be those having cysteine, homocysteine or any other thiol-containing amino acid residue as one or more moieties constituting the same, whose thiol group(s) are protected with the protecting group(s), and one of typical examples of those peptides is glutathione. The present method is further applicable for preparation of amino acids or peptides having disulfide linkage(s) (e.g. cystine, oxytocin, va~opressin, somatostatin, insulin, calcitonine, trypsin inhibitor, ribonuclease or lysozyme). The peptides employable in the present method may have moieties other than amino acid residues so far as the peptides have the protected thiol group(s).
As examples of said mercuric salt of carboxylic acid may be mentioned the mercuric salts of acetic acid, . .
.: , .
- ..:
dichloroacetlc acld, trichloroacetic acld, trifluoroacetlc ~cid, the mercuric salts of acetic acid and trifluoroaGe~ic ~cid being particularly preferred. Based on th~ amino scid or peptide, such a mercurie salt may be used in a propQrtion of, for example, 1 to about 4 molar eq~ivalents.
In tbe practice of this invention, the dlrect addltl~n of fiuch a mercuric salt to the reaction system ma~ be replaced by the additi~n of materials whlch are able to yield 9uch a mercurlc salt in the reaction sy~em.
For example, mercurlc oxide may be dlssolved ln said carb~xylic acid (I) so that the resultant mercuric salt may be utilized for the purposes of this inventlon.
The method o~ thls lnvention may normally be practiced ln the presence of a solvent. The solvent may for example be carboxylic aclds (I) (e.g. acetic acid, dichloroacetic acid, trichloroacetlc acid or trlfluoroacetlc acid or a mixture of such an acid wlth water. It ls also posslble to select one, whlch ls able to dissolve the deslred compound wlth comparatlve ease and is suited to the contemplated reactlon, from among such other solvents as water, alcohols (e.g. methanol, ethanol, propanol, butanol, and mixtures thereof with water), tetrahydrofuran, dioxane, dlmethylformamide, chloroform, acetonitrile, formic acid and mixtures thereof with water, for instance. The method of this invent~on may be practiced at an appropriate temperature, for example in the range of about -20C to about 100C, preferably about 0C to about 80C.
While the treating time accordlng to this invention depends upon such factors as the types and amounts Or the amino acid or peptide,solvent and mercuric salt employed, it is in many cases sufficient to conduct the treatment for about 30 seconds to 12 hours.
While the thiol-protecting group, i.e. p-methoxybenzyl, l-adamantyl, t-butyl, may thus be removed by the method Or this lnvention, the amino acid or peptide may be in the form of a mercaptide in certain cases and the free thiol group may be regenerated by a procedure known per se. For example, the mercaptide is dissolved or suspended in water, an aqueous solvent or a solvent (e.g.
formic acid, acetic acid, dimethylformamlde, tetrahydrofuran) and treated with a mercapto compound (e.g. hydrogen sulfide, mercaptoethanol, thioglycolic acid, sodium sulfide, ammonium sulfide), whereby the free thiol group is regenerated.
The method of this invention has the following utility features.
(1) The removal of protective groups is accomplished under extremely mild conditions.
Method ~or removal of thlol-protecting groups SPECIFICATION
This invention relates to a novel and useful method for removal of thiol-protecting groups.
More specifically, the present invention is directed to a method for removal o~ protective group(s) from an amino acid or peptide havlng thiol group(s) protected with p-methoxybenzyl, l-adamantyl or t-butyl.
Generally, in organic reactions it is often necessary to protect and, then, regenerate a thiol group. Especially in peptide synthesis, protection and regeneration of thiol groups are quite important.
Among procedures of protecting a thiol group and, then, removing the protective group, most widely available procedures are in these days the procedure of protectlng lt with a benzyl group and, then, removing this protective group wlth metallic sodium in liquid ammonia and of protecting the thiol with p-methoxybenzyl and then removing the latter with refluxing tri-fluoroacetic acid or anhydrous hydrogen fluoride ["J. Biol. Chem. Vol. 108, 753-761 (1935)", Bull. Chem.
Soc. Japan, Vol. 37, No. 39 433-434 (1964)", and "Bull.
Chem. Soc. Japan, Vol. 40, 2164-2167 ~1967)"~.
However, because the condit~ons employed in the removal .; -.. . ~
.
107~300 of such protective groups are somewhat drastic, there are cases in which it is found to be impossible to selectively remove the thiol-protecting group with the other functional groups and protective groups being kept intact.
Because t-butyl and l-adamantyl groups may be easily introduced to the thiol group, attempts have been made to utilize them as protective groups for thiol, only to find, however, that these protective groups are very difficult to remove subsequently ["J. A. C. S. Vol.
8~, 201-207 (1963)"]. Thus, no effective procedure for removing such protective groups has been known.
Under these circumstances we sought for a new method of regenerating a thiol group protected by p-methoxybenzyl, l-adamantyl or t-butyl and have discovered that such protective groups may be removed with extreme ease and in good yield by treating the substrate under very mild con~itions, i.e. with a mercuric salt of a carboxylic acid of the formula CX3COOH (I) wherein X is hydrogen, chlorine or fluorine.
Developed as a sequel of the above finding, this invention relates to a method for removal of a protective group from an amino acid or peptide having thiol group(s) protected with p methoxybenzyl, l-adamantyl or t-butyl characterized in that the protected amino acid or peptide is treated with a mercuric salt of a ~, .
1~7430~
carboxylic acid (I).
In accordance with the invention~ an amino acid or peptide having a thiol group or thiol groups protected with p-methoxybenzyl, l-adamantyl or t-butyl is treated with a mercuric salt of a carboxylic acid (I).
The present method is applicable to any amino acids having thiol group(s) protected with the protective group(s) and those amino acids may be exemplified by cysteine or homocysteine. The present method ls also applicable to any peptides having thiol group(s) protected with the protective group(s).
These peptides may be those having cysteine, homocysteine or any other thiol-containing amino acid residue as one or more moieties constituting the same, whose thiol group(s) are protected with the protecting group(s), and one of typical examples of those peptides is glutathione. The present method is further applicable for preparation of amino acids or peptides having disulfide linkage(s) (e.g. cystine, oxytocin, va~opressin, somatostatin, insulin, calcitonine, trypsin inhibitor, ribonuclease or lysozyme). The peptides employable in the present method may have moieties other than amino acid residues so far as the peptides have the protected thiol group(s).
As examples of said mercuric salt of carboxylic acid may be mentioned the mercuric salts of acetic acid, . .
.: , .
- ..:
dichloroacetlc acld, trichloroacetic acld, trifluoroacetlc ~cid, the mercuric salts of acetic acid and trifluoroaGe~ic ~cid being particularly preferred. Based on th~ amino scid or peptide, such a mercurie salt may be used in a propQrtion of, for example, 1 to about 4 molar eq~ivalents.
In tbe practice of this invention, the dlrect addltl~n of fiuch a mercuric salt to the reaction system ma~ be replaced by the additi~n of materials whlch are able to yield 9uch a mercurlc salt in the reaction sy~em.
For example, mercurlc oxide may be dlssolved ln said carb~xylic acid (I) so that the resultant mercuric salt may be utilized for the purposes of this inventlon.
The method o~ thls lnvention may normally be practiced ln the presence of a solvent. The solvent may for example be carboxylic aclds (I) (e.g. acetic acid, dichloroacetic acid, trichloroacetlc acid or trlfluoroacetlc acid or a mixture of such an acid wlth water. It ls also posslble to select one, whlch ls able to dissolve the deslred compound wlth comparatlve ease and is suited to the contemplated reactlon, from among such other solvents as water, alcohols (e.g. methanol, ethanol, propanol, butanol, and mixtures thereof with water), tetrahydrofuran, dioxane, dlmethylformamide, chloroform, acetonitrile, formic acid and mixtures thereof with water, for instance. The method of this invent~on may be practiced at an appropriate temperature, for example in the range of about -20C to about 100C, preferably about 0C to about 80C.
While the treating time accordlng to this invention depends upon such factors as the types and amounts Or the amino acid or peptide,solvent and mercuric salt employed, it is in many cases sufficient to conduct the treatment for about 30 seconds to 12 hours.
While the thiol-protecting group, i.e. p-methoxybenzyl, l-adamantyl, t-butyl, may thus be removed by the method Or this lnvention, the amino acid or peptide may be in the form of a mercaptide in certain cases and the free thiol group may be regenerated by a procedure known per se. For example, the mercaptide is dissolved or suspended in water, an aqueous solvent or a solvent (e.g.
formic acid, acetic acid, dimethylformamlde, tetrahydrofuran) and treated with a mercapto compound (e.g. hydrogen sulfide, mercaptoethanol, thioglycolic acid, sodium sulfide, ammonium sulfide), whereby the free thiol group is regenerated.
The method of this invention has the following utility features.
(1) The removal of protective groups is accomplished under extremely mild conditions.
(2) The protective groups may be removed in good yield.
, ' . ' ~,, . ,. . . ,: '. .
, ' . ' ~,, . ,. . . ,: '. .
(3) The procedure and after-treatment are easy and simple.
(4) The protective group on thiol may be selectively removed wlth other functional and protective groups being kept intact.
The following are ex~mplary processes for the production of the amino acid or peptide with protected-thiol group(s) as reference examples and exemplary procedures for removing the protective groups as working examples.
Throughout this specification, when abbreviations are used to denote amino acids, peptides, protective groups, etc., the nomenclature prescribed by IUPAC IUB
and the trivial names commonly used in this particular field will be employed. Whenever any amino acid has D-and L-configurations, any reference to such an acid indicates the L-form thereof unless the D-form is specifically mentioned.
Reference Example 1 Adm Production of S-l-adamantyl-L-cysteine (H-Cys-OH) In 120 mQ of trifluoroacetic acid is dissolved 12.1 g of L-cysteine, followed by addition of 15.2 g of 1-adamantanol. The mixture is stirred at room temperature for 12 hours. The trifluoroacetic acid is distilled off under reduced pressure. The oily residue is dissolved in 200-mQ of water and cooled wi~h ice. The solution , , 10743()0 is then brought to pH 6.0 with concentrated aqueous ammonia, whereupon crystals separate out. The crystals are collected by filtration, rinsed with water, ethanol and ether, and recrystallized from water.
Yield: 23.0 g; m.p.: 227.0-228.0C (decomp.) [a~D6-14.5(c=0.51, glacial acetic acid) Elemental analysis:
Calcd. for C13H22o2N3 C, 60.90; H, 8.65; N, 5.46; S, 12.51 Found C, 59.51; H, 8.16; N, 5.41; S, 12.33 Reference Example 2 tBu Production of S-t-butyl-L-cysteine (H-Cys-OH) Using L~cysteine and t-butanol, the same procedure as Reference Example 1 is followed to produce the above compound. m.p.: 244.0-245.0C
[~]D6-15.4(c=0.57; glaclal acetic acid) Elemental analysis:
Calcd. for C7H15O2NS
C, 45.13; H, 8.65~ N, 7.52; S, 17.22 Found C, 45.11; H, 8.32; N, 7.47; S, 17.77 Reference Example 3 Production of t-butoxycarbonyl-S-t-butyl-L-cysteine t-Bu dicyclohexylamine salt (Boc-Cys-OH-DCHA) tBu In the conventional manner, the H-Cys-OH obtained in Reference Example 2 is t-butoxycarbonylated with t-10743~0 butyl-S-4, 6-dimethylpyrimidine-2-ylthiolcarbonate and, then, crystallized as the dicyclohexylamine salt.
Yield: 90%; m.p.: 181.0-182.5C
[a]D +7-4 (c=0.83, methanol~
Elemental analysis:
Calcd. for C24~46O4N2S
C, 62.93; H, 10.10; N, 6.10; S, 6.99;
Found C, 62.92; H, 10.36; N, 5.95; S, 7.05 t-Butoxycarbonyl-S-l-adamantyl-L-cysteine Adm dicyclohexylamine salt (Boc-cys-oH-DcHA) i p the same manner as above.
Yield: 71.4%; m.p.: 173.0-174.0C;
[a]D5+5.8 (c=0.55, methanol) Elemental analysis:
Calcd- for C30H53O4N2S
C, 67.00; H, 9.93; N, 5.21; S, 5.96 Found C, 67.09; H, 9.88; N, 5.08; S, 5.85 The t-butoxycarbonyl-S-p-methoxybenzyl-L-cysteine PMB
Boc-Cys OH) used is a commercial product.
Reference Example 4 tBu Production of Boc-Gly-Cys-Gly-OMe tBu (1) Production of Boc-Cys-Bly-OMe In 30 mQ of chloroform is dissolved 3.0 g of glycine methyl ester hydrochloride and, under ice-cooling, 3.36 mQ of triethylamine is added. To this 10~4300 mlxture is added the N-hydroxy-5-norbornene-2,3-tBu icarboximide (HONB) ester of Boc-Cys-OH (prepared tBu from 9. 2 g of Boc-Cys-OH-DCHA and 3.6 g of HONB in dioxane with 4.1 g of dicyclohexylcarbodiimide as a condensin~ agent) and the mixture is stirred at room temperature for 12 hours. The chloroform is distilled off under reduced pressure and the oily residue is dissolved in 24-150 mQ of acetic acid and washed with a 4% aqueous solution of sodium hydrogen carbonate, a saturated aqueous solution of citric acid and, finally, water. The ethyl acetate is then evaporated off and the residue is crystallized ~y addition of water.
ield: 5.9 g.~ m.p.: 73.0-75.0C.
~a]26-17.9 (c=0.56, dimethylformamide) lemental analysis:
c . 15H28 5 2S
c, 51.70, H, 8.10; N, 8.043 S, 9.20 Found C, 51.70; H, 8.12; N, 8.05~ S, 9.14 tBu (2) ProductiOn of z_Gly-cys-Gly-oMe In 30 mQ of trifluoroacetic acid is disæolved 5.23 g tBu f Boc Cys Gly OMe and the solution is shaken at room temperature for 15 minutes. The trifluoroacetic acid is evaporated off undér reduced pressure and the residue is dried well. It is then dissolved in 30 mQ of d~methylformamide and, under ice-cooling, 7.0 mQ of 1~74300 triethylamine is added. Following addition of 5.8 g of Z-Gly-ONB, the mixture is stirred for 4~ hours. The dimethylformamide is evaporated off under reduced pressure and the residue is dissolved in ethyl acetate.
The solution is washed with a 4% aqueous solution of sodium hydrogen carbonate, lN-hydrochloric acid and water, followed by concentration to dryness under reduced pressure. To the residue is added petroleum ether and the mixture is allowed to stand, whereupon crystals separate out. The crystals are collected by filtration.
Yield: 6.1 g., m.p.: undeterminable because it is liquid at room temperature.
[a]D6-11.4(c=0.46, dimethylformamide) Elemental analysis:
Calcd. for C20H2906N3S
C, 54.65~ H, 6.65; N, 9.56; S, 7.30 Found C, 55.11; H, 6.60; N, 9.48; S, 6.55 PMB
Z-Gly-Cys-Gly ~Me is also produced in the same manner as above.
m.p.: 90.0-95.0Cj [~]D7-23.1 (c=0.55; dimethylformamide) Elemental analysis:
Calcd- for C24H297N3S
C, 57.20; H, 5.809 N, 8.34; S, 6.37 Found C, 57.60~ H, 5.81, N, ~.23; S, 6.36 Example 1 Recovery of cysteine from S-protected cysteine (1) S-protected cysteine (lmM) is dissolved in 10 mQ of trifluoroacetic acid, followed by addition of 0.2 m~ of anisole. The mixture is cooled to 0C and 319 mg (lmM) of Hg(CH3C00)2 is added. The mixture is stirred at 0C for 15 minutes. The trifluoroacetic acid is evaporated off under reduced pressure over a period of 5 minutes and 40 mQ of water is added to the residue. The mixture is washed once with ether.
Hydrogensulfide gas is bubbled into this solution for 30 minutes and the resultant HgS is filtered off, followed by concentration of the filtrate. This residue is dissolved in small amount of water, followed by evaporation of water to remove the excess of the acid.
This procedure is repeated three times and the final residue is analyzed by an amino acid analyzer. The results are shown in the following table. S-Benzyl group is quite stable under the above conditions.
! ' .
I S-protected ' tBu , Adm PMB
1~ amino ac~d I H-Cys-OH ~ H-Cys-OH H-Cys-OH
~ !
cysteine 1 99.8% 102.0% 100 ~ ¦
~, ! . ~I, .
No removal of protective groups takes place when the above r~action is conducted using the acetate of Ag, Cu, Zn, Ni or Pb instead of Hg(CH3COO)2.
(2) S-protected cysteine (lmM) is dissolved in 10 mQ of 80% formic acid or 80% acetic acid, followed by addition of 0.2 mQ of anisole. At room temperature, 319 mg (lmM) of Hg(CH3COO)2 is added and the mixture is stirred for 1 hour. The reaction mixture is treated and analyzed in the same manner as (1).
The results are set forth in the following table.
\ S-protected mino acidPMB tBu Adm \ H-Cys-OH H-Cys-OH H-Cys-OH
Solvent \
80% formic acld 85.0% 86.0% 80.0%
80% acetic acid 73.0% 48.0% _ (3) S-protected cysteine (lmM) is dissolved in 20 mQ of 80% acetic acid, followed by addition of 0.2 mQ
of anisole and, then, ~12 mg (1.2mM) of Hg(C~3COO)2.
The mixture is stirred at room temperature for 1 hour.
The reaction mixture is then treated and analyzed in the same manner as (1).
. . -. . . .
107~300 The results are shown below.
S-protected PMB tBu amino acid H-Cys-OH H-Cys-OH
.
% Recovery 94.0% 92.0%
When a solution of Hg(CCQ3COO)2, instead of Hg(C~3COO)2, is used, the percent recovery of cysteine PMB
from H-Cys-OH is 96.0%.
Example 2 Production of Z-Gly-Cys-Gly-OMe PMB
(1) From Z-Gly-Cys-Gly-OMe:
In 45 mQ of cold trifluoroacetic acid is dissolved PMB
3.02 g of Z-Gly-Cys-Gly-OMe, followed by addltion of 1.5 mQ of anisole. Following addition of 1.91 g of Hg(CH3CO'O)2 at 0C, the mlxture is stirred for 15 minutes.
The trifluoroacetic acid is distilled off under reduced pressure and ether is added to the residue. The resultant precipitate is collected by filtration.
Yield: 4.1 g.
Elemental analysis:
Calcd. for Cl~H20O8N3SF3Hg C, 31.069 H, 2.90j N, 6.04; S, 4.61; Hg, 28.82;
F. 8.19 .. ~ ..
.. , ~ - .
:' .''~` . :..... ..
~074300 Found C, 31.02; ~, 2.87; N, 6.18; S, 4.60; Hg, 28.68;
F, 8.18 In 30 mQ of dimethylformamide is dissolved 2.0 g of the above mercaptide9 and H2S gas is bubbled into the solution for 80 minutes. The resultant HgS is removed by filtration with Celite as à filter aid and the flltrate is concentrated to dryness under reduced pressure. To the crystalline residue is added water and the crystals are recovered by filtration and recrystallized from methanol and water.
Yield: 1.1 g (100%); m.p.: 130-132C
[a]26-5.7 (c=0.57; dimethylformamide) Elemental analysis:
Calcd- for C16H216N3S
C, 50.12; H, 5.52j N, 10.96; S, 8.36 Found C, 49.61; H, 5.18; N, 10.97; S, 8.44 tBu (2) From Z-Gly-Cys-Gly-OMe:
tBu Treatment of Z-Gly-Cys-Gly-OMe in the same manner as (1) gives the mercaptide and Z-Gly-Cys-Gly-OMe in the yields o~ 84.2% and 84.5%, respectively.
In~melting point and ~]D~ this compound is identical with the compound obtained in (1).
Elemental analysis:
Calcd. C, 50.12; H, 5.52; N, 10.96~ S, 8.36 Found C, 50.13j H, 5.61j N, 10.77; S, 8.27 (33 The table below shows the yields of Z-Gly-Cys-Gly-OMe crystals as produced by the treatment Or PMB
Z-Gly-Cys-Gly-OMe with Hg(CF3COO)2 in 80% acetic acid.
In 20 mQ of 80% acetic acid is diss~lved 1 mM
PMB
Z-Gly-Cys_Gly_oMe~ followed by the addition of 512 mg (1.2 mM) or 853 mg(2.0 mM) of Hg(CF3C00)2. The mixture is stirred at room temperature for 60 minutes.
Following addition of 40 mQ of 80% acetic acid, H2S
gas is bubbled into the mixture over 20 minutes. The HgS is filtered off and the filtrate is concentrated to dryness under reduced pressure. To the residue is added water and the resultant crystals are collected by filtration and recrystallized from ethyl acetate.
He(CF3C)2 1.2 molar equiv. 2.0 molar equiv.
Yield 83.9% 76.5%
When the above reaction procedure is carried out by using 1.2 ~olar e~uivalents of ~g(CC13CO~2 in lieu o~
Hg(CF3COO)2, the yield of Z-Gly~Cys-Gly-OMe is 74.9%.
Example 3 Production of bis-t-butoxycarbonyl-L-cystine In 200 mQ of 80% aqueous methanol is dissolved Pr~B
3.41 g f BOC Cys OH and, following addition of 5.12 g ' 10'~4300 of Hg(C~3C00)2, the solution is stirred at room temperature for 2 hours. Then, following addition of 100 mQ of 80% aqueous methanol, H2S gas is bubbled into the reaction mixture for 30 minutes. The resultant HgS is filtered off and the filtrate is concentrated under reduced pressure to half its initial volume.
To the residue is added 4.2 ~ of sodium hydro~en carbonate and the mixture is further concentrated under reduced pressure. After the methanol has been removed, 100 mQ of water is added to the residue, and oxidation is carried out by the introduction of air over a period of 40 hours. The mixture is acidified with citric acid and the resultant oil is extracted twice with 150 mQ portions of ethyl acetate. The ethyl acetate is distilled off, and the residue is crystallized by addition of petroleum ether and is collected by filtration. The crystalline product is recrystallized from ethyl acetate.
Yield: 1.85 g., m.p.: 149.0-150.0C (decomp.) [~]D -136.4 (c=1.84, in methanol) Elemental analysis:
Calcd. for C16H2808N2S2 C, 43.623 H1 6.48; N, 6.363 S, 14.55 Found C, 43.72; H, 6.343 N, 6.24; S, 14.25 Example 4 Production of oxytocin 10'~4300 (1) Production of Boc-Cts(PMB)-Tyr-Ile-Gln-Asn-Cys (PMB)-Pro-Leu-Gly-resin Boc-Gly-resin (Gly content 2.2 mM) is placed in the reaction vessel of a Shimadzu automatic peptide synthesizer APS-800 and the following procedure is carried out in sequence.
(1) Washing with dichloromethane (2 min. x 3 times) (2) 50% trifluoroacetic acid-dichloromethane (10 min. x twice) (3) Washing with dichloromethane (2 min. x 3 times) (4) Washing with ethanol (2 min. x 3 times)
The following are ex~mplary processes for the production of the amino acid or peptide with protected-thiol group(s) as reference examples and exemplary procedures for removing the protective groups as working examples.
Throughout this specification, when abbreviations are used to denote amino acids, peptides, protective groups, etc., the nomenclature prescribed by IUPAC IUB
and the trivial names commonly used in this particular field will be employed. Whenever any amino acid has D-and L-configurations, any reference to such an acid indicates the L-form thereof unless the D-form is specifically mentioned.
Reference Example 1 Adm Production of S-l-adamantyl-L-cysteine (H-Cys-OH) In 120 mQ of trifluoroacetic acid is dissolved 12.1 g of L-cysteine, followed by addition of 15.2 g of 1-adamantanol. The mixture is stirred at room temperature for 12 hours. The trifluoroacetic acid is distilled off under reduced pressure. The oily residue is dissolved in 200-mQ of water and cooled wi~h ice. The solution , , 10743()0 is then brought to pH 6.0 with concentrated aqueous ammonia, whereupon crystals separate out. The crystals are collected by filtration, rinsed with water, ethanol and ether, and recrystallized from water.
Yield: 23.0 g; m.p.: 227.0-228.0C (decomp.) [a~D6-14.5(c=0.51, glacial acetic acid) Elemental analysis:
Calcd. for C13H22o2N3 C, 60.90; H, 8.65; N, 5.46; S, 12.51 Found C, 59.51; H, 8.16; N, 5.41; S, 12.33 Reference Example 2 tBu Production of S-t-butyl-L-cysteine (H-Cys-OH) Using L~cysteine and t-butanol, the same procedure as Reference Example 1 is followed to produce the above compound. m.p.: 244.0-245.0C
[~]D6-15.4(c=0.57; glaclal acetic acid) Elemental analysis:
Calcd. for C7H15O2NS
C, 45.13; H, 8.65~ N, 7.52; S, 17.22 Found C, 45.11; H, 8.32; N, 7.47; S, 17.77 Reference Example 3 Production of t-butoxycarbonyl-S-t-butyl-L-cysteine t-Bu dicyclohexylamine salt (Boc-Cys-OH-DCHA) tBu In the conventional manner, the H-Cys-OH obtained in Reference Example 2 is t-butoxycarbonylated with t-10743~0 butyl-S-4, 6-dimethylpyrimidine-2-ylthiolcarbonate and, then, crystallized as the dicyclohexylamine salt.
Yield: 90%; m.p.: 181.0-182.5C
[a]D +7-4 (c=0.83, methanol~
Elemental analysis:
Calcd. for C24~46O4N2S
C, 62.93; H, 10.10; N, 6.10; S, 6.99;
Found C, 62.92; H, 10.36; N, 5.95; S, 7.05 t-Butoxycarbonyl-S-l-adamantyl-L-cysteine Adm dicyclohexylamine salt (Boc-cys-oH-DcHA) i p the same manner as above.
Yield: 71.4%; m.p.: 173.0-174.0C;
[a]D5+5.8 (c=0.55, methanol) Elemental analysis:
Calcd- for C30H53O4N2S
C, 67.00; H, 9.93; N, 5.21; S, 5.96 Found C, 67.09; H, 9.88; N, 5.08; S, 5.85 The t-butoxycarbonyl-S-p-methoxybenzyl-L-cysteine PMB
Boc-Cys OH) used is a commercial product.
Reference Example 4 tBu Production of Boc-Gly-Cys-Gly-OMe tBu (1) Production of Boc-Cys-Bly-OMe In 30 mQ of chloroform is dissolved 3.0 g of glycine methyl ester hydrochloride and, under ice-cooling, 3.36 mQ of triethylamine is added. To this 10~4300 mlxture is added the N-hydroxy-5-norbornene-2,3-tBu icarboximide (HONB) ester of Boc-Cys-OH (prepared tBu from 9. 2 g of Boc-Cys-OH-DCHA and 3.6 g of HONB in dioxane with 4.1 g of dicyclohexylcarbodiimide as a condensin~ agent) and the mixture is stirred at room temperature for 12 hours. The chloroform is distilled off under reduced pressure and the oily residue is dissolved in 24-150 mQ of acetic acid and washed with a 4% aqueous solution of sodium hydrogen carbonate, a saturated aqueous solution of citric acid and, finally, water. The ethyl acetate is then evaporated off and the residue is crystallized ~y addition of water.
ield: 5.9 g.~ m.p.: 73.0-75.0C.
~a]26-17.9 (c=0.56, dimethylformamide) lemental analysis:
c . 15H28 5 2S
c, 51.70, H, 8.10; N, 8.043 S, 9.20 Found C, 51.70; H, 8.12; N, 8.05~ S, 9.14 tBu (2) ProductiOn of z_Gly-cys-Gly-oMe In 30 mQ of trifluoroacetic acid is disæolved 5.23 g tBu f Boc Cys Gly OMe and the solution is shaken at room temperature for 15 minutes. The trifluoroacetic acid is evaporated off undér reduced pressure and the residue is dried well. It is then dissolved in 30 mQ of d~methylformamide and, under ice-cooling, 7.0 mQ of 1~74300 triethylamine is added. Following addition of 5.8 g of Z-Gly-ONB, the mixture is stirred for 4~ hours. The dimethylformamide is evaporated off under reduced pressure and the residue is dissolved in ethyl acetate.
The solution is washed with a 4% aqueous solution of sodium hydrogen carbonate, lN-hydrochloric acid and water, followed by concentration to dryness under reduced pressure. To the residue is added petroleum ether and the mixture is allowed to stand, whereupon crystals separate out. The crystals are collected by filtration.
Yield: 6.1 g., m.p.: undeterminable because it is liquid at room temperature.
[a]D6-11.4(c=0.46, dimethylformamide) Elemental analysis:
Calcd. for C20H2906N3S
C, 54.65~ H, 6.65; N, 9.56; S, 7.30 Found C, 55.11; H, 6.60; N, 9.48; S, 6.55 PMB
Z-Gly-Cys-Gly ~Me is also produced in the same manner as above.
m.p.: 90.0-95.0Cj [~]D7-23.1 (c=0.55; dimethylformamide) Elemental analysis:
Calcd- for C24H297N3S
C, 57.20; H, 5.809 N, 8.34; S, 6.37 Found C, 57.60~ H, 5.81, N, ~.23; S, 6.36 Example 1 Recovery of cysteine from S-protected cysteine (1) S-protected cysteine (lmM) is dissolved in 10 mQ of trifluoroacetic acid, followed by addition of 0.2 m~ of anisole. The mixture is cooled to 0C and 319 mg (lmM) of Hg(CH3C00)2 is added. The mixture is stirred at 0C for 15 minutes. The trifluoroacetic acid is evaporated off under reduced pressure over a period of 5 minutes and 40 mQ of water is added to the residue. The mixture is washed once with ether.
Hydrogensulfide gas is bubbled into this solution for 30 minutes and the resultant HgS is filtered off, followed by concentration of the filtrate. This residue is dissolved in small amount of water, followed by evaporation of water to remove the excess of the acid.
This procedure is repeated three times and the final residue is analyzed by an amino acid analyzer. The results are shown in the following table. S-Benzyl group is quite stable under the above conditions.
! ' .
I S-protected ' tBu , Adm PMB
1~ amino ac~d I H-Cys-OH ~ H-Cys-OH H-Cys-OH
~ !
cysteine 1 99.8% 102.0% 100 ~ ¦
~, ! . ~I, .
No removal of protective groups takes place when the above r~action is conducted using the acetate of Ag, Cu, Zn, Ni or Pb instead of Hg(CH3COO)2.
(2) S-protected cysteine (lmM) is dissolved in 10 mQ of 80% formic acid or 80% acetic acid, followed by addition of 0.2 mQ of anisole. At room temperature, 319 mg (lmM) of Hg(CH3COO)2 is added and the mixture is stirred for 1 hour. The reaction mixture is treated and analyzed in the same manner as (1).
The results are set forth in the following table.
\ S-protected mino acidPMB tBu Adm \ H-Cys-OH H-Cys-OH H-Cys-OH
Solvent \
80% formic acld 85.0% 86.0% 80.0%
80% acetic acid 73.0% 48.0% _ (3) S-protected cysteine (lmM) is dissolved in 20 mQ of 80% acetic acid, followed by addition of 0.2 mQ
of anisole and, then, ~12 mg (1.2mM) of Hg(C~3COO)2.
The mixture is stirred at room temperature for 1 hour.
The reaction mixture is then treated and analyzed in the same manner as (1).
. . -. . . .
107~300 The results are shown below.
S-protected PMB tBu amino acid H-Cys-OH H-Cys-OH
.
% Recovery 94.0% 92.0%
When a solution of Hg(CCQ3COO)2, instead of Hg(C~3COO)2, is used, the percent recovery of cysteine PMB
from H-Cys-OH is 96.0%.
Example 2 Production of Z-Gly-Cys-Gly-OMe PMB
(1) From Z-Gly-Cys-Gly-OMe:
In 45 mQ of cold trifluoroacetic acid is dissolved PMB
3.02 g of Z-Gly-Cys-Gly-OMe, followed by addltion of 1.5 mQ of anisole. Following addition of 1.91 g of Hg(CH3CO'O)2 at 0C, the mlxture is stirred for 15 minutes.
The trifluoroacetic acid is distilled off under reduced pressure and ether is added to the residue. The resultant precipitate is collected by filtration.
Yield: 4.1 g.
Elemental analysis:
Calcd. for Cl~H20O8N3SF3Hg C, 31.069 H, 2.90j N, 6.04; S, 4.61; Hg, 28.82;
F. 8.19 .. ~ ..
.. , ~ - .
:' .''~` . :..... ..
~074300 Found C, 31.02; ~, 2.87; N, 6.18; S, 4.60; Hg, 28.68;
F, 8.18 In 30 mQ of dimethylformamide is dissolved 2.0 g of the above mercaptide9 and H2S gas is bubbled into the solution for 80 minutes. The resultant HgS is removed by filtration with Celite as à filter aid and the flltrate is concentrated to dryness under reduced pressure. To the crystalline residue is added water and the crystals are recovered by filtration and recrystallized from methanol and water.
Yield: 1.1 g (100%); m.p.: 130-132C
[a]26-5.7 (c=0.57; dimethylformamide) Elemental analysis:
Calcd- for C16H216N3S
C, 50.12; H, 5.52j N, 10.96; S, 8.36 Found C, 49.61; H, 5.18; N, 10.97; S, 8.44 tBu (2) From Z-Gly-Cys-Gly-OMe:
tBu Treatment of Z-Gly-Cys-Gly-OMe in the same manner as (1) gives the mercaptide and Z-Gly-Cys-Gly-OMe in the yields o~ 84.2% and 84.5%, respectively.
In~melting point and ~]D~ this compound is identical with the compound obtained in (1).
Elemental analysis:
Calcd. C, 50.12; H, 5.52; N, 10.96~ S, 8.36 Found C, 50.13j H, 5.61j N, 10.77; S, 8.27 (33 The table below shows the yields of Z-Gly-Cys-Gly-OMe crystals as produced by the treatment Or PMB
Z-Gly-Cys-Gly-OMe with Hg(CF3COO)2 in 80% acetic acid.
In 20 mQ of 80% acetic acid is diss~lved 1 mM
PMB
Z-Gly-Cys_Gly_oMe~ followed by the addition of 512 mg (1.2 mM) or 853 mg(2.0 mM) of Hg(CF3C00)2. The mixture is stirred at room temperature for 60 minutes.
Following addition of 40 mQ of 80% acetic acid, H2S
gas is bubbled into the mixture over 20 minutes. The HgS is filtered off and the filtrate is concentrated to dryness under reduced pressure. To the residue is added water and the resultant crystals are collected by filtration and recrystallized from ethyl acetate.
He(CF3C)2 1.2 molar equiv. 2.0 molar equiv.
Yield 83.9% 76.5%
When the above reaction procedure is carried out by using 1.2 ~olar e~uivalents of ~g(CC13CO~2 in lieu o~
Hg(CF3COO)2, the yield of Z-Gly~Cys-Gly-OMe is 74.9%.
Example 3 Production of bis-t-butoxycarbonyl-L-cystine In 200 mQ of 80% aqueous methanol is dissolved Pr~B
3.41 g f BOC Cys OH and, following addition of 5.12 g ' 10'~4300 of Hg(C~3C00)2, the solution is stirred at room temperature for 2 hours. Then, following addition of 100 mQ of 80% aqueous methanol, H2S gas is bubbled into the reaction mixture for 30 minutes. The resultant HgS is filtered off and the filtrate is concentrated under reduced pressure to half its initial volume.
To the residue is added 4.2 ~ of sodium hydro~en carbonate and the mixture is further concentrated under reduced pressure. After the methanol has been removed, 100 mQ of water is added to the residue, and oxidation is carried out by the introduction of air over a period of 40 hours. The mixture is acidified with citric acid and the resultant oil is extracted twice with 150 mQ portions of ethyl acetate. The ethyl acetate is distilled off, and the residue is crystallized by addition of petroleum ether and is collected by filtration. The crystalline product is recrystallized from ethyl acetate.
Yield: 1.85 g., m.p.: 149.0-150.0C (decomp.) [~]D -136.4 (c=1.84, in methanol) Elemental analysis:
Calcd. for C16H2808N2S2 C, 43.623 H1 6.48; N, 6.363 S, 14.55 Found C, 43.72; H, 6.343 N, 6.24; S, 14.25 Example 4 Production of oxytocin 10'~4300 (1) Production of Boc-Cts(PMB)-Tyr-Ile-Gln-Asn-Cys (PMB)-Pro-Leu-Gly-resin Boc-Gly-resin (Gly content 2.2 mM) is placed in the reaction vessel of a Shimadzu automatic peptide synthesizer APS-800 and the following procedure is carried out in sequence.
(1) Washing with dichloromethane (2 min. x 3 times) (2) 50% trifluoroacetic acid-dichloromethane (10 min. x twice) (3) Washing with dichloromethane (2 min. x 3 times) (4) Washing with ethanol (2 min. x 3 times)
(5) Washing with chloroform (2 min. x 3-times)
(6) 10% triethylamine-chloroform ( 2 min. & 10 min., once each)
(7) Washing with chloroform (2 min. x 3 times)
(8) The condensation reaction with 6.6 mM of BOC-amino acid symmetric anhydride (60 min.) or 6.6 mM of BOC-amino acid p-nitrophenyl ester (12 hours for BOC-Asn, Boc-Gl~ or Boc-Tyr)
(9) Washing with dichloromethane (2 min. x 3 times)
(10) Acetylation of the unreacted amino group (4.5%
acetic anhydride-dichloromethane) tll) Washing with dichloromethane (2 min. x 3 times) After all the reaction cycles have been completed, the product is washed with acetic acid, dimethylformamide and methanol in that order and dried. Yield: 13.15 g (2) Production of Boc-Cys(PMB)-Tyr-Ile-Gln-Asn-Cys(PMB)-Pro-Leu-Gly-~2 In 70 mQ of methanol containing 15.5% of ammonia is suspended 12.6 g of the resin obtained in (1) and the suspension is stirred at room temperature for 46 hours.
The resin is filtered off and washed twice with 20 mQ
of dimethylformamide. The filtrate and washings are pooled and concentrated to dryness under reduced pressure. To the residue is added 50 mQ of ether and the resultant powder is collected by filtration and dried. Yield: 2.86 g. This product is purified by repeated reprecipitation from hot methanol, aqueous ethanol and dimethylformamide-ethanol.
Yield: 1.0 g.
This powder is developed on a 2.8 x 4.0 cm column of silica gel (solvent system: chloroform-methanol-water-pyridine-acetic acid=1085 : 150 : 25 : 63 : 84).
The fractions rich in the desired compound are pooled and concentrated to dryness.
Yield: 768 mg.
[~]21~39.1 (c=0.47, in dimethylformamide) Elemental analysis:
r 66H980l6Nl2s2cH3cooH H20 C, 55.529 H, 6.92; N, 11.78g S9 4.49 Found Cg 55.24; H, 7.Q0; N, il.92; S, 4.43 ~074300 t3) Production of oxytocin In 1 mQ of trifluoroacetic acid is dissolved 125 mg of the powder produced in (2) together with 0.22 mQ of anisole, followed by the addition of 127 mg of Hg(CH3C00)2. The mixture is allowed to stand at room temperature for 30 minutes. Following addition of 30 mQ of ether, the resultant precipitate is collected by filtration (173 mg). This precipitate is dissolved in 3 mQ of 50% aqueous acetic acid and, following addition of 0.31 mQ of 2-mercaptoethanol, the resultant precipitate is filtered off. The filtrate is concentrated to dryness under reduced pressure. The residue is dis~olved in 1 mQ of 50% aqueous acetic acid and the solution is passed into a column (2.2. x 92.5 cm) of Sephadex G~15, elution being carried out with 50%
acetic acid. The fractions 149 to 188 mQ are pooled and lyophilized (85 mg).
The lyophilizate is dissolved in 150 mQ of 0.1%
aqueous acetic acid and after the solution is ad~usted to pH 7.3 with lN-aqueous ammonia, air is bubbled into the solution. After 4.5 hours~ the solution is ad~usted to pH 3.5 and lyophilized (76 mg). The lyophilizate is developed on a column similar to that mentioned above. The fractions from 154 to 188 mQ are collected and lyophilized to give 50 mg of oxytocin.
[a]22-24.6(c=0.47, lN-aqueous solution of acetic acid) . .
10'74;~00 Amino acid analysis- Asp 1.03, Glu 1.13, Pro 1.06, Gly 1.00~ Cys 1.97, Ile 1.03, Leu 1.03, Tyr 0.90 Thin-layer chromatography: Rf(n butanol-acetic acid-water =3:1:1)=0.44; Rf(n-butanol-ethyl acetate-acetic acid-water=l:l:l:l)=0.66~ (a single spot each).Example 5 Production of Somatostatin PMB
(1) Synthesis of H-Cys-OtBu~L-tartrate PMB
To a suspension of 4.82 g (0.02 M) of H-Cys-OH
in 150 mQ of dioxane is added dropwise 4.8 mQ of con-centrated sulfuric acid under stirring at room temperature.
Into the mixture is bubbled 75 mQ of isobutylene under ice-cooling. The mixture is tightly sealed and left standing at room temperature for 12 hours. The mixture is poured on to 300 mQ of lN solution of sodium hydroxide and extracted three times with ether. The ether layer is dried over anhydrous sodium sulfate and distilled off under reduced pressure. The oily residue is dissolved in 50 mQ of ether, followed by addition of 3 mQ of 6.69N hydrochloric acid-dioxane. The resulting crystals are collected by filtration and recrystallized from ethyl acetate. Yield: 2.4 g.
In ether is suspended 1.99 g of the crystals and 10 mQ of lN solution of sodium hydroxide is added.
The mixture is shaken well enough to become a solution ~074300 and extracted twice with ether. The ether solu~ion is combined, dried and distilled under reduced pressure.
The oily residue is dissolved in ethanol. In the solution is dissolved 900 mg of L-tartaric acid and the solution is left standing for a while. Resulting crystals are collected by filtration and recrystallized twice from ethanol.
Yield: 2.3 g m.p. 100.0-102.0C.
[]D7-3~2 (c=0.53, dimethylformamide) Elemental analysis:
Calcd. for C15H23O3NS C4H66 / 2 C, 49.99; H, 6.62~ N3 3.07; S, 7.02 Found C, 49.85; H, 6.74; N~ 2.989 S, 6.68 (2) Synthesis of Z-Thr-Ser-OMe In 50 mQ of dimethylformamide is dissolved 6.22 g (0.04 M) of H-Ser-OMe-HCQ, followed by addition of trlethylamine under ice-cooling. Resulting salt is removed by filtration and 16.6 g (0.04 M) of Z-Thr-ONB
is added to the filtrate. After the mixture is stirred at room temperature for 12 hours, the insolubles are removed by filtration and the filtrate is concentrated in vacuo. The oily residue is dissolved in ethyl acetate and saturated with NaCQ and washed with 1 N HCQ
and 5% NaHCO3. After being dried, the ethyl acetate ~s distilled off and resulting crystals are collected.
Yield: 10.6 g (74.5%) m.p. 133.0-135.0C;
~3D3+10.1 (c=0.86, dimethylformamide) Elemental analysis:
Calcd. for C16H227N2 C9 54.23; H, 6.26; N, 7.91 Found C~ 54.44~ H, 6.33; N, 7.84 (3) Synthesis of Z-Phe-Thr-Ser-OMe In 100 mQ of methanol is dissolved 9.91 g (0.028 M) of Z-Thr-Ser OMe, foilowed by addition of 6.3 mQ of 6.69 N HCQ-dioxane. The mixture is sub~ected to catalytic reduction in the presence of palladium black in a conventional manner. The catalyst is removed by filtration and the filtrate is concentrated. The residue is dissolved in 50 m~ of dimethylformamide and the solution is neutralized with 4.31 mQ of triethylamine, followed by filtration. To the filtrate is added Z-Phe-ONB, which is prepared from 8.38 g (0.028 M) of Z-Phe-OH, 6.0 g (0.034 M) of HONB and 5.78 g (0.028 M) of dicyclohexylcarbodiimide, and the mixture is stirred at room temperature for 12 hours. The solvent is distilled off in vacuo, and the residue is dissolved in ethyl acetate, followed by washing and drying in a conventional manner. Evaporation of the solvent separates out crystals. After being cooled, the crystals are collected by filtration.
Yield: 11.0 g (80.0%). m.p.: 180.0-182.0C.
[~]23-3.8O (c=0.679 dimethylformamide) :, Elemental analysis:
Calcd- for C25H318N3-1/2H2 C, 58.81; H, 6.12; N, 8.23 Found C, 58.80j H9 6.25j N, 8.26 (4) Synthesis of Z(NO2)-Thr-OH DCHA
The compound is prepared by the conventional procedure [F,H. Carpenter, D.T. Gish, J. Amer. Chem. Soc., 74, 3818 (1952)] with NaHCO3 as a base.
Yield: 50.0% m.p.: 186.0-18~.0C.
[a]D3+14.9 (c=0.77, dimethylformamlde~
Elemental analysis:
Calcd. for C12~I14N27 C12H23 C, 60.11; H, 7.78; N, 8.76 Found C3 60.14; H, 7.83; N, 8.80 (5) Synthesis of Z(N02)-Thr-Phe-Thr-Ser-OMe In a conventional procedure, 11.0 g (0.022 M) of Z-Phe-Thr-Ser-OMe is catalytically reduced into H-Phe-Thr-Ser-OMe in methanol. To this is added a tetrahydrofuran solution of Z(NO2)-Thr-ONB which is prepared from 10.6 g (0.022 M) of Z(NO2)-Thr-OH DCHA, 3.94 g (0.022 M) of HONB and 4.54 g (0.022 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours. The solvent is distilled off, followed by addition of water. Resulting crystals are collected by filtration and recrystallized from methanol.
.
.: .
,..... .
Yield: 12.8 g (90.1%). m.p.: 194.0-196.0C
[a]23-0.7 (c=0.72, dimethylformamide)`
Elemental analysis:
Calcd- for C29H3712N5 c, 53.789 H, 5.76; N, 10.81 Found C, 53.80j H, 5.85; N, 10.75 (6) Synthesis of Z(NO2)-Thr-Phe-Thr-Ser-NHNH2 In 100 mQ of dimethylformamide is dissolved 12.3 (0.019 M) of Z(N02)-Thr-Phe-Thr-Ser-OMe, followed by addition of 100 mQ of methanol and further addition of 9.5 mQ (0.19 M) of hydrazine hydrate. The mixture is left standing at room temperature for 4 8 hours and cooled with ice. Resulting crystals are collected by filtration and recrystallized from methanol.
Yield: 11.4 g (92.7%). m.p.: 229.0-232.0c (decomp.) [a ] 23+ 3.2 ( c =o . 44, dimethylformamide) Elemental analysis:
Calcd. for c28H37ollN7 H20 c, 50.529 H, 5.913 N, 14.81 Found C, 50.89; H, 5.929 N, 14.90 PMB
7) Synthesis of Z(N02)-Thr-Phe-Thr~Ser-Cys-0 Bu In 50 mQ of dimethylformamide is dissol~Ted 3.24 g 0.005 M) of Z(N02)-Thr-Phe-Thr-Ser-NHNH2. While the ' solution is cooled to -20c, 3.1 mQ of 6.51 N HCQ-; dioxane is added and 0.81 mQ (o.006 M) isoamylnitrite .
~ ~74300 is added dropwise. After the minutes, the mixture is ~urther cooled to -50C and neutralized with triethylamine.
PMB
Meanwhile, 2.24 g (0.005 M) f H Cys otBU
L-tartrate is dissolved in dimethylformamide and the solution is neutralized with 1.4 mQ of triethylamine under ~ce-cooling. This solution is combined with the azide solution prepared as above and the combined solution is stirred at 4C for 48 hours. Insolubles are removed by filtration and the filtrate is concentrated.
Water is added to the residue and the resulting powder is collected by filtration. The powder is recrystallized twice from methanolO
Yield: 3.55 ~ (77.7%). m.p.: 193.0-195.0C (de~omp.) [a]25-18.0 (c=0.56, dimethylformamide) Elemental analysis: ' -; Calcd. ~or C43H5614N6$
C, 56.57; H, 6.18~ N, 9.21~ S, 3.51 Found C, 56.313 H, 6.o8; N, 9.34; S, 3.47(8) Synthesis of Z(NO2)-Trp-OH
The compound is prepared by a conventional procedure.
Yield: 88.6% m.p.: 108.0-115.0C (decomp.) ~a]D3-43.5C (c=0.65, dimethylformamide~
Elemental analysis:
Calcd- for Cl9H174N3 2H2 C, 58.91; H, 5.46; N, 10.85 Found C, 59.639 H, 4.63; N, 10.74 - 2~ -: . , .,~
BOC
(9) Synthesis of Z(N02)-Trp-~ys-OH DCHA
In 100 mQ of dimethylformamide is dissolved BOC
) H-Lys-OH9 followed by addition of 4.2 m~
(0.03 M) of triethylamine. To the solution is added Z(N02)-Trp-ONB which is prepared from 10.5 g (0.03 M) of Z(NO2)-Trp-OH, 5.4 g (0.03 M) of HONB and 6.19 g (0.03 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours and the solvent is distilled off. After addition of 30 mQ
of lN HCQ, the mixture is dissolved in ethyl acetate.
The ethyl acetate layer is washed three times with water, dried and distilled to evaporate the solvent.
The oily residue is dissolved in 100 mQ of acetonitrile and 6.0 mQ (0.03 M) of dicyclohexylamine is added and the mixture is left standing in a refrigerator.
Resulting gelatinous crystals are collected by flltration ,;
".;
and recrystallized from acetonitrile.
Yield: 12.5 g (52.6%) m.p.: 95.0-100.0C.
[a]25-20.9 (c=0.56, dimethylformamide) Elemental analysis:
Calcd. for C30~37gNs C12H23 Cg 63.61; H, 7.63~ N, 10.60 Found C, 63.779 H9 7.67; N~ 11.08 BOC PMB
(10) Synthesis of Z(NO2)-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OtBu In 150 mQ of a mixture of acetic acid and water (8:2 by volume), is dissolved 3.29 ~ (0.0036 M) ., ,,, ;
PMB
of Z(NO2)-Thr-Phe-Thr-Ser-Cys-OtBu and the solution is subjected to catalytic reduction at 50C for 8 hours in the presence of palladium black. The catalyst is removed by filtration and the filtrate is lyophilized.
The lyophilizate is dissolved in methanol and ether is added. Resulting precipitate is collected by filtration and reprecipitated from methanol-ether. Yield: 2.75 g In a small amount of dimethylformamide is dissolved 2.62 g (0.0033 M) of the powder obtained above. A solut,ion of 627 mg of p-toluenesulfonic acid in dimethylformamide is added to the solution under cooling and the mixture is ~ left standing. Most of the dimethylformamide is distilled - off and ether is added to the residue. Resulting powder is collected by filtration and dissolved in 20 mQ of dimethylformamide, followed by addition of Z(NO2)-BOC
Trp-Lys-OH~ which is prepared from 2.88 g (o.oo36 M) of ; BOC
Z(NO2)-Trp-Lys-OH~DCHA, and further addition of 1.30 g (0.0072 M) of HONB, 0.42 mQ (0.0033 M) of N-ethylmorpholine and dicyclohexylcarbodiimide 819 mg (0.004 M). The mixture ls stirred at -10C for 3 hours and further stirred at room temperature for 48 hours. Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated. Water is added to the residue.
Resulting precipitate is collected by filtration , . .
107430(~
and recrystallized twice from methanol.
Yleld: 3.5 g (80.6%) m.p.: 183.0-185.0C (decomp.) [~]D5_26.60 (c=0.67, dimethylformamide) Elemental analysis:
Calcd. for C64H84l8N10S
C, 58.52; H, 6.459 N, 10.66~ S, 2.44 Found C, 58.36; H, 6.47, N~ 10.49; S, 2.54
acetic anhydride-dichloromethane) tll) Washing with dichloromethane (2 min. x 3 times) After all the reaction cycles have been completed, the product is washed with acetic acid, dimethylformamide and methanol in that order and dried. Yield: 13.15 g (2) Production of Boc-Cys(PMB)-Tyr-Ile-Gln-Asn-Cys(PMB)-Pro-Leu-Gly-~2 In 70 mQ of methanol containing 15.5% of ammonia is suspended 12.6 g of the resin obtained in (1) and the suspension is stirred at room temperature for 46 hours.
The resin is filtered off and washed twice with 20 mQ
of dimethylformamide. The filtrate and washings are pooled and concentrated to dryness under reduced pressure. To the residue is added 50 mQ of ether and the resultant powder is collected by filtration and dried. Yield: 2.86 g. This product is purified by repeated reprecipitation from hot methanol, aqueous ethanol and dimethylformamide-ethanol.
Yield: 1.0 g.
This powder is developed on a 2.8 x 4.0 cm column of silica gel (solvent system: chloroform-methanol-water-pyridine-acetic acid=1085 : 150 : 25 : 63 : 84).
The fractions rich in the desired compound are pooled and concentrated to dryness.
Yield: 768 mg.
[~]21~39.1 (c=0.47, in dimethylformamide) Elemental analysis:
r 66H980l6Nl2s2cH3cooH H20 C, 55.529 H, 6.92; N, 11.78g S9 4.49 Found Cg 55.24; H, 7.Q0; N, il.92; S, 4.43 ~074300 t3) Production of oxytocin In 1 mQ of trifluoroacetic acid is dissolved 125 mg of the powder produced in (2) together with 0.22 mQ of anisole, followed by the addition of 127 mg of Hg(CH3C00)2. The mixture is allowed to stand at room temperature for 30 minutes. Following addition of 30 mQ of ether, the resultant precipitate is collected by filtration (173 mg). This precipitate is dissolved in 3 mQ of 50% aqueous acetic acid and, following addition of 0.31 mQ of 2-mercaptoethanol, the resultant precipitate is filtered off. The filtrate is concentrated to dryness under reduced pressure. The residue is dis~olved in 1 mQ of 50% aqueous acetic acid and the solution is passed into a column (2.2. x 92.5 cm) of Sephadex G~15, elution being carried out with 50%
acetic acid. The fractions 149 to 188 mQ are pooled and lyophilized (85 mg).
The lyophilizate is dissolved in 150 mQ of 0.1%
aqueous acetic acid and after the solution is ad~usted to pH 7.3 with lN-aqueous ammonia, air is bubbled into the solution. After 4.5 hours~ the solution is ad~usted to pH 3.5 and lyophilized (76 mg). The lyophilizate is developed on a column similar to that mentioned above. The fractions from 154 to 188 mQ are collected and lyophilized to give 50 mg of oxytocin.
[a]22-24.6(c=0.47, lN-aqueous solution of acetic acid) . .
10'74;~00 Amino acid analysis- Asp 1.03, Glu 1.13, Pro 1.06, Gly 1.00~ Cys 1.97, Ile 1.03, Leu 1.03, Tyr 0.90 Thin-layer chromatography: Rf(n butanol-acetic acid-water =3:1:1)=0.44; Rf(n-butanol-ethyl acetate-acetic acid-water=l:l:l:l)=0.66~ (a single spot each).Example 5 Production of Somatostatin PMB
(1) Synthesis of H-Cys-OtBu~L-tartrate PMB
To a suspension of 4.82 g (0.02 M) of H-Cys-OH
in 150 mQ of dioxane is added dropwise 4.8 mQ of con-centrated sulfuric acid under stirring at room temperature.
Into the mixture is bubbled 75 mQ of isobutylene under ice-cooling. The mixture is tightly sealed and left standing at room temperature for 12 hours. The mixture is poured on to 300 mQ of lN solution of sodium hydroxide and extracted three times with ether. The ether layer is dried over anhydrous sodium sulfate and distilled off under reduced pressure. The oily residue is dissolved in 50 mQ of ether, followed by addition of 3 mQ of 6.69N hydrochloric acid-dioxane. The resulting crystals are collected by filtration and recrystallized from ethyl acetate. Yield: 2.4 g.
In ether is suspended 1.99 g of the crystals and 10 mQ of lN solution of sodium hydroxide is added.
The mixture is shaken well enough to become a solution ~074300 and extracted twice with ether. The ether solu~ion is combined, dried and distilled under reduced pressure.
The oily residue is dissolved in ethanol. In the solution is dissolved 900 mg of L-tartaric acid and the solution is left standing for a while. Resulting crystals are collected by filtration and recrystallized twice from ethanol.
Yield: 2.3 g m.p. 100.0-102.0C.
[]D7-3~2 (c=0.53, dimethylformamide) Elemental analysis:
Calcd. for C15H23O3NS C4H66 / 2 C, 49.99; H, 6.62~ N3 3.07; S, 7.02 Found C, 49.85; H, 6.74; N~ 2.989 S, 6.68 (2) Synthesis of Z-Thr-Ser-OMe In 50 mQ of dimethylformamide is dissolved 6.22 g (0.04 M) of H-Ser-OMe-HCQ, followed by addition of trlethylamine under ice-cooling. Resulting salt is removed by filtration and 16.6 g (0.04 M) of Z-Thr-ONB
is added to the filtrate. After the mixture is stirred at room temperature for 12 hours, the insolubles are removed by filtration and the filtrate is concentrated in vacuo. The oily residue is dissolved in ethyl acetate and saturated with NaCQ and washed with 1 N HCQ
and 5% NaHCO3. After being dried, the ethyl acetate ~s distilled off and resulting crystals are collected.
Yield: 10.6 g (74.5%) m.p. 133.0-135.0C;
~3D3+10.1 (c=0.86, dimethylformamide) Elemental analysis:
Calcd. for C16H227N2 C9 54.23; H, 6.26; N, 7.91 Found C~ 54.44~ H, 6.33; N, 7.84 (3) Synthesis of Z-Phe-Thr-Ser-OMe In 100 mQ of methanol is dissolved 9.91 g (0.028 M) of Z-Thr-Ser OMe, foilowed by addition of 6.3 mQ of 6.69 N HCQ-dioxane. The mixture is sub~ected to catalytic reduction in the presence of palladium black in a conventional manner. The catalyst is removed by filtration and the filtrate is concentrated. The residue is dissolved in 50 m~ of dimethylformamide and the solution is neutralized with 4.31 mQ of triethylamine, followed by filtration. To the filtrate is added Z-Phe-ONB, which is prepared from 8.38 g (0.028 M) of Z-Phe-OH, 6.0 g (0.034 M) of HONB and 5.78 g (0.028 M) of dicyclohexylcarbodiimide, and the mixture is stirred at room temperature for 12 hours. The solvent is distilled off in vacuo, and the residue is dissolved in ethyl acetate, followed by washing and drying in a conventional manner. Evaporation of the solvent separates out crystals. After being cooled, the crystals are collected by filtration.
Yield: 11.0 g (80.0%). m.p.: 180.0-182.0C.
[~]23-3.8O (c=0.679 dimethylformamide) :, Elemental analysis:
Calcd- for C25H318N3-1/2H2 C, 58.81; H, 6.12; N, 8.23 Found C, 58.80j H9 6.25j N, 8.26 (4) Synthesis of Z(NO2)-Thr-OH DCHA
The compound is prepared by the conventional procedure [F,H. Carpenter, D.T. Gish, J. Amer. Chem. Soc., 74, 3818 (1952)] with NaHCO3 as a base.
Yield: 50.0% m.p.: 186.0-18~.0C.
[a]D3+14.9 (c=0.77, dimethylformamlde~
Elemental analysis:
Calcd. for C12~I14N27 C12H23 C, 60.11; H, 7.78; N, 8.76 Found C3 60.14; H, 7.83; N, 8.80 (5) Synthesis of Z(N02)-Thr-Phe-Thr-Ser-OMe In a conventional procedure, 11.0 g (0.022 M) of Z-Phe-Thr-Ser-OMe is catalytically reduced into H-Phe-Thr-Ser-OMe in methanol. To this is added a tetrahydrofuran solution of Z(NO2)-Thr-ONB which is prepared from 10.6 g (0.022 M) of Z(NO2)-Thr-OH DCHA, 3.94 g (0.022 M) of HONB and 4.54 g (0.022 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours. The solvent is distilled off, followed by addition of water. Resulting crystals are collected by filtration and recrystallized from methanol.
.
.: .
,..... .
Yield: 12.8 g (90.1%). m.p.: 194.0-196.0C
[a]23-0.7 (c=0.72, dimethylformamide)`
Elemental analysis:
Calcd- for C29H3712N5 c, 53.789 H, 5.76; N, 10.81 Found C, 53.80j H, 5.85; N, 10.75 (6) Synthesis of Z(NO2)-Thr-Phe-Thr-Ser-NHNH2 In 100 mQ of dimethylformamide is dissolved 12.3 (0.019 M) of Z(N02)-Thr-Phe-Thr-Ser-OMe, followed by addition of 100 mQ of methanol and further addition of 9.5 mQ (0.19 M) of hydrazine hydrate. The mixture is left standing at room temperature for 4 8 hours and cooled with ice. Resulting crystals are collected by filtration and recrystallized from methanol.
Yield: 11.4 g (92.7%). m.p.: 229.0-232.0c (decomp.) [a ] 23+ 3.2 ( c =o . 44, dimethylformamide) Elemental analysis:
Calcd. for c28H37ollN7 H20 c, 50.529 H, 5.913 N, 14.81 Found C, 50.89; H, 5.929 N, 14.90 PMB
7) Synthesis of Z(N02)-Thr-Phe-Thr~Ser-Cys-0 Bu In 50 mQ of dimethylformamide is dissol~Ted 3.24 g 0.005 M) of Z(N02)-Thr-Phe-Thr-Ser-NHNH2. While the ' solution is cooled to -20c, 3.1 mQ of 6.51 N HCQ-; dioxane is added and 0.81 mQ (o.006 M) isoamylnitrite .
~ ~74300 is added dropwise. After the minutes, the mixture is ~urther cooled to -50C and neutralized with triethylamine.
PMB
Meanwhile, 2.24 g (0.005 M) f H Cys otBU
L-tartrate is dissolved in dimethylformamide and the solution is neutralized with 1.4 mQ of triethylamine under ~ce-cooling. This solution is combined with the azide solution prepared as above and the combined solution is stirred at 4C for 48 hours. Insolubles are removed by filtration and the filtrate is concentrated.
Water is added to the residue and the resulting powder is collected by filtration. The powder is recrystallized twice from methanolO
Yield: 3.55 ~ (77.7%). m.p.: 193.0-195.0C (de~omp.) [a]25-18.0 (c=0.56, dimethylformamide) Elemental analysis: ' -; Calcd. ~or C43H5614N6$
C, 56.57; H, 6.18~ N, 9.21~ S, 3.51 Found C, 56.313 H, 6.o8; N, 9.34; S, 3.47(8) Synthesis of Z(NO2)-Trp-OH
The compound is prepared by a conventional procedure.
Yield: 88.6% m.p.: 108.0-115.0C (decomp.) ~a]D3-43.5C (c=0.65, dimethylformamide~
Elemental analysis:
Calcd- for Cl9H174N3 2H2 C, 58.91; H, 5.46; N, 10.85 Found C, 59.639 H, 4.63; N, 10.74 - 2~ -: . , .,~
BOC
(9) Synthesis of Z(N02)-Trp-~ys-OH DCHA
In 100 mQ of dimethylformamide is dissolved BOC
) H-Lys-OH9 followed by addition of 4.2 m~
(0.03 M) of triethylamine. To the solution is added Z(N02)-Trp-ONB which is prepared from 10.5 g (0.03 M) of Z(NO2)-Trp-OH, 5.4 g (0.03 M) of HONB and 6.19 g (0.03 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours and the solvent is distilled off. After addition of 30 mQ
of lN HCQ, the mixture is dissolved in ethyl acetate.
The ethyl acetate layer is washed three times with water, dried and distilled to evaporate the solvent.
The oily residue is dissolved in 100 mQ of acetonitrile and 6.0 mQ (0.03 M) of dicyclohexylamine is added and the mixture is left standing in a refrigerator.
Resulting gelatinous crystals are collected by flltration ,;
".;
and recrystallized from acetonitrile.
Yield: 12.5 g (52.6%) m.p.: 95.0-100.0C.
[a]25-20.9 (c=0.56, dimethylformamide) Elemental analysis:
Calcd. for C30~37gNs C12H23 Cg 63.61; H, 7.63~ N, 10.60 Found C, 63.779 H9 7.67; N~ 11.08 BOC PMB
(10) Synthesis of Z(NO2)-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OtBu In 150 mQ of a mixture of acetic acid and water (8:2 by volume), is dissolved 3.29 ~ (0.0036 M) ., ,,, ;
PMB
of Z(NO2)-Thr-Phe-Thr-Ser-Cys-OtBu and the solution is subjected to catalytic reduction at 50C for 8 hours in the presence of palladium black. The catalyst is removed by filtration and the filtrate is lyophilized.
The lyophilizate is dissolved in methanol and ether is added. Resulting precipitate is collected by filtration and reprecipitated from methanol-ether. Yield: 2.75 g In a small amount of dimethylformamide is dissolved 2.62 g (0.0033 M) of the powder obtained above. A solut,ion of 627 mg of p-toluenesulfonic acid in dimethylformamide is added to the solution under cooling and the mixture is ~ left standing. Most of the dimethylformamide is distilled - off and ether is added to the residue. Resulting powder is collected by filtration and dissolved in 20 mQ of dimethylformamide, followed by addition of Z(NO2)-BOC
Trp-Lys-OH~ which is prepared from 2.88 g (o.oo36 M) of ; BOC
Z(NO2)-Trp-Lys-OH~DCHA, and further addition of 1.30 g (0.0072 M) of HONB, 0.42 mQ (0.0033 M) of N-ethylmorpholine and dicyclohexylcarbodiimide 819 mg (0.004 M). The mixture ls stirred at -10C for 3 hours and further stirred at room temperature for 48 hours. Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated. Water is added to the residue.
Resulting precipitate is collected by filtration , . .
107430(~
and recrystallized twice from methanol.
Yleld: 3.5 g (80.6%) m.p.: 183.0-185.0C (decomp.) [~]D5_26.60 (c=0.67, dimethylformamide) Elemental analysis:
Calcd. for C64H84l8N10S
C, 58.52; H, 6.459 N, 10.66~ S, 2.44 Found C, 58.36; H, 6.47, N~ 10.49; S, 2.54
(11) Synthesls of Z-Phe-Phe~OtBu According to a conventional procedure, 17.8 g (0.05 M) of Z~Phe-OtBu is catalytically reduced in methanol to H-Phe--O Bu. To th~s is added a solution of Z-Phe-ONB which is prepared from 15.0 g tO 05 M) of Z-Phe OH, 9.0 g (0.05 M) of E~ONB and 10.3 g 3 (0.05 M) of dicyclohexylcarbodiimide, and the mixture is stirred at room temperature for 12 hours. The dioxane is distilled off and the residue is dissolved in ethyl acetate. The solution is washed in a conventional manner, dried and distilled to evaporate the ethyl acetate. Resulting crystals are collected by filtration and recrystallized from ethyl acetate.
Yield: 15.4 g (60.0%) m.p.: 107.0-109.0C
[a]D7-13.5 (c=0 53~ dimethylformamide) Elemental analysis:
- Calcd. ~or C30H345N2 C, 71.96~ H, 6.82; N, 5.57 Found C9 71.61~ H, 6.64~ N, 5.50 .i . , ,, :~ .
: '; . '':' ~12) Synthesis of BOC-Asn-Phe-Phe-OtBu In a conventional procedure, 13.1 g (0.026 M) of Z-Phe-Phe-OtBu is catalytically reduced in methanol to H-Phe-Phe-OtBu. This is dissolved in 50 mQ of dimethyl-formamide together with 6.o4 g (0.026 M) of BOC-Asn-Oi~ and 4.65 g (0.026 M) of I~ONB. Under ice-cooling, 5.36 g (0.026 M) of dicyclohexylcarbodiimide is added and the mixture is stirred at 0C for 3 hours and at room temperature for further 12 hours. Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo, followed by addition of water.
Resulting crystals are collected by filtration and recrystallized twice from methanol.
Yield: 11.0 g (72.8%). m.p.: 163.0-166.0C
~a]D7-28.9 (c=0.85, dimethylformamide) Elemental analysis:
Calcd- for C31H427N4 C, 63.905 H, 7.27; N, 9.62 Found C, 64.12; H~ 7.20~ N, 9~90 BOC
(13) Synthesis of Z-Lys-Asn-Phe-Phe-OH
In 100 mQ of trifluoroacetic acid is dissolved 10.5 g (0.018 M) of BOC-Asn-Phe-Phe-O Bu and the mixture is left standing at room temperature for 1.5 hours.
The trifluoroacetic acid ~s distilled off under reduced pressure and ether is added to the residue. Resulting :' ~074300 preclpitate 's collected by f ltratlon and dried and dissolved in 100 mQ of dimethylformam de; To the solutlon is added 5.04 mQ (0.036 M) of triethylamine and a BOC
Z-Lys-ONB which is prepared from 8.2 g BOC
(0.0216 M) of Z-Lys-OH, 3.8 g (0.0216 M) of HONB and 4.4 g (0.0216 M3 of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours, followed by addition of 10 mQ of acetic acid. The solvent is distilled off in vacuo and water is added to the residue. Resulting precipitate is collected by filtration and purified by reprecipitating twice from aqueous methanol and then from dimethylformamide-ethyl acetate.
Yield: 11.1 g (78.1%) m.p.: 194.0-196.0C (decomp.) [~]27_26.9O (c=0.59, dimethylformamide) Elemental analysis:
Calcd. for C41H5210N6 C, 62.42; H, 6.64; N, 10.65 Found C, 62.27; H, 6.66; N, 10.72 PMB
(14) Synthesis of Z(NO2)-Cys-OH
The compound is prepared by a conventional procedure.
Yield: 80.0% m.p.: 163.0-165.0C.
~a]23-51.2 ~c=0.61~ dimethylformamide) Elemental analysis: Calcd. for ClgH20O7N2S
Gj 54.28; H, 4.79; N, 6.66; S, 7.62 ~ 30 ~
'.`' : :
Found C, 54.18; H, 4.74; N, 6.579 S, 7.56 PMB BOC
(15) Synthesis of Z(NO2)-Cys-Lys-Asn~Phe-Phe-OH
According to a conventional procedure, 10.3 g ~OC
Z-Lys-Asn-phe-phe-oH is catalytically educed in a mixture of acetic acid and water (8:2 by BOC
volume). Resulting H Lys Asn Phe Phe-OH is purified by two times of reprecipitation from ethanol.
Yield: 7.0 g In 100 mQ of dimethylformamide is suspended 6.55 g (0.01 M) of the compound, followed by addition of 1.40 mQ
0.01 M) of triethylamine and further addition of a PMB
solution of dimethylformamide in Z(NO ) Cys ONB which , ~ ~ PMB
is prepared from 5.05 g (0.012 M) of Z(NO2)-Cys-OH~
2.15 g (0.012 M) of HONB and 2.48 g (0.012 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours. After additlon of 10 mQ
of acetic acid, the dimethylformamide is distilled off in vacuo and water is added ~o the res~due. Resulting precipitate is collected by filtration and recrystallized twice from a mixture of methanol and water (8:2 by volume) Yield: 9.6 g (92.3~) m.p.: 209.0-210.0C (decomp.) ~a~27-24.6 (c-0.74, dimethylformamide) Elemental analysis:
Calcd- for C52H64O14N8S
C, 59.08; H, 6.10, N, 10.60; S, 3.03 Found C, 59.043 H9 6.02; Ng 10.60; S, 3.19 (16) Synthesis of BOC-Ala-Gly-OBzl In 50 mQ of dimethylformamide is dissolved 8.10 g (0.024 M) of H-Gly-OBzl-p-Ts-OlI ~nd under ice-cooling 3.36 mQ (0.024 M) of triethylamine is added. To this is added ~OC-Ala-ONB which is prepared from 4.35 g (0.023 M) of BOC-Ala-OH, 4.12 g (0.023 M) of HONB
and 4.74 g (0.023 M) of dicyclohexylcarbodiimide.
The mixture is stirred at room temperature for 12 hours and distilled off in vacuo. The residue is dissolved in ethyl acetate, washed in a conventional manner and dried. The ethyl acetate is dlstilled off and petroleum ether is added to the residue to crystallize it. The crystals are collected by filtration and recrystallized from ethyl acetate-petroleum ~enzin.
Yield: 5.5 g (71.2%) m.p.: X6.0-87.0C.
[~]27-10.8 (c=0.56, dimethylformamide) Elemental analysis:
Calcd. for C17H245N2 C, 60.70; H, 7.19; N, 8.33 Fo~md C, 60.829 H, 7~19a N, 3.28 (17) Synthesis of BOC-Ala-Gly-OH
According to a conventional procedure, the compound is produced by reducing catalytically 5.29 g (0.015 M) of BOC-Ala-Gly-OBzl in methanol.
- . ~' ' '',''~''' '"
~0743 Yield: 3.3 g (85.3%) m.p.: 111.0-113.0C (decomp.) [~]D7 6.8 (c=0.56, dimethylformamide) Elemental analysis:
C, 47.05; H, 7.50; N, 10.98 Found C~ 46.66; H, 7.65; N, 11.03 PMB BOC
(18) Synthesis of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-OH
In a mixture of acetic acid and water (~:2 by PMB BOC
volume)g 5.2 g (C.005 M) of z(No2)-cys-Lys-Asn-phe-Phe-OH is catalytically reduced on palladium black at 50C for 8 hours. The catalyst is removed by filtration and the filtrate is concentrated and the residue is triturated with ether. The resulting powder is collected by filtration and reprecipitated from ethanol.
Yield: 4.05 g The powder is suspended in 50 mQ of dimethylformamide, followed by addition of 0.7 mQ of triethylamine and further addition of BOC-Ala-Gly~ONB which is prepared from 1.48 g (0.006 ~) of BOC-Ala-Gly-OH, 1.08 g (0.006 M) of HONB and 1.24 g (0.006 M) of dicyclohexylcarbodiimide.
The mixture is vigorously stirred for 12 hours. After being acidified with 10 mQ of acetic acid3 the dimethylformamide ls distilled off in vacuo. Water is added to the residueg and the resulting powder is collected by filtration and reprecipitated twice 10'74300 from a mixture of methanol and water (8:2 by volume).
Yield 3.5 g (60.4p) m.p.: 201.0-203.0C (decomp.) []D5-28.2O (c=0.69g dimethylformamide) Elemental analysis:
Calcd. for C54H75O14N9 Cg 58.639 H, 6.83; N, 11.409 Sg 2.90 F'ound C, 58.86, H, 6.~3; N, 11.539 S, 2.79 PMB BOC
(19) Synthesis of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-BOC PMB
Lys-Thr-Phe-Thr-Ser-Cys-OtBu In 150 mQ of a mixture of acetic acid and water 8:2 by volume) is suspended 3.29 g (0.0025 M) of BOC PMB
Z(NO2)-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OtBu and the compound is catalytically reduced on palladium black at 50C
for 5 hours. The catalyst is removed by filtration and the filtrate is concentrated under reduced pressure.
A small amount of ethanol is added to the residue and ether is further added to triturate the residue. This procedure is repeated.
Yield: 2.90 g In a small amount of dimethylformamide is dissolved 2.6~ g (0.0023 M) of the compound prepared above, and under ice-cooling a solution of 437 mg (0.0023 M) of p-toluenesulfonic acid in dimethylformamide is added dropwise. After the mlxture is left standing for a whileg the solvent is distilled off in vacuo and ether ` ' ' '' , ~'"~ ''.. '.' .' ~ , ;
~074300 is added to trlturate the residue; Re~ulting powder is dissolved in 20 mQ of dimethylformamide, followed by addition of 823 mg (0.0046 M) of HONB, 0.29 mQ
(0.0023 M) of N-ethylmorpholine and 2.54 g (0.0023 M) PMB BOC
of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-OH. At -10C, 949 mg (0.0046 M) of dicyclohexylc~rboaiimide in dimethylformamide is added and the mixture is stirred for 3 hours and for further 72 hours at room temperature.
Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo. Water is added to the residue, and resulting precipitate is collected by filtration and reprecipitated twice from methanol.
Yield: 3.8 g (75.1%) mOp.: 224.0-226.0C (decomp.) [a]25-18.3 (c=0.70, dimethylformamide) Elemental analysis:
Calcd. for Clo9Hl54o26Nl8s2 - C, 59.62; H, 7.069 N, 11.479 S, 2.92 Found Cg 59.559 Hg 6.759 N, 11.32; S, 2.95 (20) Synthesis of H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (somatostatin) In 20 mQ of à mixture of acetic acid and water (8:2 by volume) is dissolved 220 mg (0.1 mM) of PMB BOC BOC
BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr Phe-PMB
Thr-Ser-Cys-OtBuO To the solution is added 171 mg ~0.4 mM) of Hg (CF3C00)2 and the mixture is stirred at - 35 ~
10`~4~(~0 room temperature for 12 hours. Hydrogen sulfide gas is bubbled into the reaction mixture and resulting black precipitate is removed by filtration with celite. The filtrate is concentrated under reduced pressure and the residue is triturated with ether containing a minor amount of ~-mercaptoethanol. Resulting powder is collected by filtration and dried. Yield: 193 mg.
The compound is dissolved in 500 mQ of dimethyl-formamide, followed by addition of 0.28 mQ of 10%
triethylamine-dimethylformamide and further addition of 30 mg of 1,2-diiodoethane. The reaction mixture is stirred at room temperature for 30 minutes and the solvent is distilled off in vacuo. The residue is trifurated with ether and resulting powder is collected by filtration and dried.
Yield: 15~ mg The compound is dissolved in 3 mQ of a mixture of trifluoroacetic acid and water (9:1 by volume) and the solution is shaken at room temperature for 30 minutes. The trifluoroacetic acid is distilled off below 30C under reduced pressure for 10 minutes.
Ether is added3 and resulting precipitate is collected by filtration and dissolved in a small amount of water.
The solution is passed through a column (2.0 x 7.0 cm) of Amberlite IRA-410 tacetate form) to effect ion exchange. The effluent of 60 m~ is passed through a - ~ , 10743~0 column (5.5 x 35.0 cm) of Sephadex LH-20, which is eluted with 0.1 N acetic acid solution. Major fractions of 560-640 mQa~e collected and lyophilized.
Yield: 20.4 mg [~]D6-31.2 (c=0.13, 1% acetic acid) Thin layer chromatography: Rf=0.60 (n-butanol:
pyridine: acetic acid: water=30 : 20 : 6 : 24 by volume, Avicel), Rf=0.40 (n-butanol: ethyl acetate:
acetic acid: water=l : 1 : 1 : 1 by volume, silica gel) Amino acid analysis (6N HCQ , 110C, 24 hours) Lys, 1.97(2); Asp, 1..01(1); Thr, 1.95(2);
Ser, 0.92(1); Gly, 1.00(1); Ala, 1.02(1);
Half Cys, 1.58(2); Phe, 3.01(3)~ average recovery ~2%.
Yield: 15.4 g (60.0%) m.p.: 107.0-109.0C
[a]D7-13.5 (c=0 53~ dimethylformamide) Elemental analysis:
- Calcd. ~or C30H345N2 C, 71.96~ H, 6.82; N, 5.57 Found C9 71.61~ H, 6.64~ N, 5.50 .i . , ,, :~ .
: '; . '':' ~12) Synthesis of BOC-Asn-Phe-Phe-OtBu In a conventional procedure, 13.1 g (0.026 M) of Z-Phe-Phe-OtBu is catalytically reduced in methanol to H-Phe-Phe-OtBu. This is dissolved in 50 mQ of dimethyl-formamide together with 6.o4 g (0.026 M) of BOC-Asn-Oi~ and 4.65 g (0.026 M) of I~ONB. Under ice-cooling, 5.36 g (0.026 M) of dicyclohexylcarbodiimide is added and the mixture is stirred at 0C for 3 hours and at room temperature for further 12 hours. Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo, followed by addition of water.
Resulting crystals are collected by filtration and recrystallized twice from methanol.
Yield: 11.0 g (72.8%). m.p.: 163.0-166.0C
~a]D7-28.9 (c=0.85, dimethylformamide) Elemental analysis:
Calcd- for C31H427N4 C, 63.905 H, 7.27; N, 9.62 Found C, 64.12; H~ 7.20~ N, 9~90 BOC
(13) Synthesis of Z-Lys-Asn-Phe-Phe-OH
In 100 mQ of trifluoroacetic acid is dissolved 10.5 g (0.018 M) of BOC-Asn-Phe-Phe-O Bu and the mixture is left standing at room temperature for 1.5 hours.
The trifluoroacetic acid ~s distilled off under reduced pressure and ether is added to the residue. Resulting :' ~074300 preclpitate 's collected by f ltratlon and dried and dissolved in 100 mQ of dimethylformam de; To the solutlon is added 5.04 mQ (0.036 M) of triethylamine and a BOC
Z-Lys-ONB which is prepared from 8.2 g BOC
(0.0216 M) of Z-Lys-OH, 3.8 g (0.0216 M) of HONB and 4.4 g (0.0216 M3 of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours, followed by addition of 10 mQ of acetic acid. The solvent is distilled off in vacuo and water is added to the residue. Resulting precipitate is collected by filtration and purified by reprecipitating twice from aqueous methanol and then from dimethylformamide-ethyl acetate.
Yield: 11.1 g (78.1%) m.p.: 194.0-196.0C (decomp.) [~]27_26.9O (c=0.59, dimethylformamide) Elemental analysis:
Calcd. for C41H5210N6 C, 62.42; H, 6.64; N, 10.65 Found C, 62.27; H, 6.66; N, 10.72 PMB
(14) Synthesis of Z(NO2)-Cys-OH
The compound is prepared by a conventional procedure.
Yield: 80.0% m.p.: 163.0-165.0C.
~a]23-51.2 ~c=0.61~ dimethylformamide) Elemental analysis: Calcd. for ClgH20O7N2S
Gj 54.28; H, 4.79; N, 6.66; S, 7.62 ~ 30 ~
'.`' : :
Found C, 54.18; H, 4.74; N, 6.579 S, 7.56 PMB BOC
(15) Synthesis of Z(NO2)-Cys-Lys-Asn~Phe-Phe-OH
According to a conventional procedure, 10.3 g ~OC
Z-Lys-Asn-phe-phe-oH is catalytically educed in a mixture of acetic acid and water (8:2 by BOC
volume). Resulting H Lys Asn Phe Phe-OH is purified by two times of reprecipitation from ethanol.
Yield: 7.0 g In 100 mQ of dimethylformamide is suspended 6.55 g (0.01 M) of the compound, followed by addition of 1.40 mQ
0.01 M) of triethylamine and further addition of a PMB
solution of dimethylformamide in Z(NO ) Cys ONB which , ~ ~ PMB
is prepared from 5.05 g (0.012 M) of Z(NO2)-Cys-OH~
2.15 g (0.012 M) of HONB and 2.48 g (0.012 M) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours. After additlon of 10 mQ
of acetic acid, the dimethylformamide is distilled off in vacuo and water is added ~o the res~due. Resulting precipitate is collected by filtration and recrystallized twice from a mixture of methanol and water (8:2 by volume) Yield: 9.6 g (92.3~) m.p.: 209.0-210.0C (decomp.) ~a~27-24.6 (c-0.74, dimethylformamide) Elemental analysis:
Calcd- for C52H64O14N8S
C, 59.08; H, 6.10, N, 10.60; S, 3.03 Found C, 59.043 H9 6.02; Ng 10.60; S, 3.19 (16) Synthesis of BOC-Ala-Gly-OBzl In 50 mQ of dimethylformamide is dissolved 8.10 g (0.024 M) of H-Gly-OBzl-p-Ts-OlI ~nd under ice-cooling 3.36 mQ (0.024 M) of triethylamine is added. To this is added ~OC-Ala-ONB which is prepared from 4.35 g (0.023 M) of BOC-Ala-OH, 4.12 g (0.023 M) of HONB
and 4.74 g (0.023 M) of dicyclohexylcarbodiimide.
The mixture is stirred at room temperature for 12 hours and distilled off in vacuo. The residue is dissolved in ethyl acetate, washed in a conventional manner and dried. The ethyl acetate is dlstilled off and petroleum ether is added to the residue to crystallize it. The crystals are collected by filtration and recrystallized from ethyl acetate-petroleum ~enzin.
Yield: 5.5 g (71.2%) m.p.: X6.0-87.0C.
[~]27-10.8 (c=0.56, dimethylformamide) Elemental analysis:
Calcd. for C17H245N2 C, 60.70; H, 7.19; N, 8.33 Fo~md C, 60.829 H, 7~19a N, 3.28 (17) Synthesis of BOC-Ala-Gly-OH
According to a conventional procedure, the compound is produced by reducing catalytically 5.29 g (0.015 M) of BOC-Ala-Gly-OBzl in methanol.
- . ~' ' '',''~''' '"
~0743 Yield: 3.3 g (85.3%) m.p.: 111.0-113.0C (decomp.) [~]D7 6.8 (c=0.56, dimethylformamide) Elemental analysis:
C, 47.05; H, 7.50; N, 10.98 Found C~ 46.66; H, 7.65; N, 11.03 PMB BOC
(18) Synthesis of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-OH
In a mixture of acetic acid and water (~:2 by PMB BOC
volume)g 5.2 g (C.005 M) of z(No2)-cys-Lys-Asn-phe-Phe-OH is catalytically reduced on palladium black at 50C for 8 hours. The catalyst is removed by filtration and the filtrate is concentrated and the residue is triturated with ether. The resulting powder is collected by filtration and reprecipitated from ethanol.
Yield: 4.05 g The powder is suspended in 50 mQ of dimethylformamide, followed by addition of 0.7 mQ of triethylamine and further addition of BOC-Ala-Gly~ONB which is prepared from 1.48 g (0.006 ~) of BOC-Ala-Gly-OH, 1.08 g (0.006 M) of HONB and 1.24 g (0.006 M) of dicyclohexylcarbodiimide.
The mixture is vigorously stirred for 12 hours. After being acidified with 10 mQ of acetic acid3 the dimethylformamide ls distilled off in vacuo. Water is added to the residueg and the resulting powder is collected by filtration and reprecipitated twice 10'74300 from a mixture of methanol and water (8:2 by volume).
Yield 3.5 g (60.4p) m.p.: 201.0-203.0C (decomp.) []D5-28.2O (c=0.69g dimethylformamide) Elemental analysis:
Calcd. for C54H75O14N9 Cg 58.639 H, 6.83; N, 11.409 Sg 2.90 F'ound C, 58.86, H, 6.~3; N, 11.539 S, 2.79 PMB BOC
(19) Synthesis of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-BOC PMB
Lys-Thr-Phe-Thr-Ser-Cys-OtBu In 150 mQ of a mixture of acetic acid and water 8:2 by volume) is suspended 3.29 g (0.0025 M) of BOC PMB
Z(NO2)-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OtBu and the compound is catalytically reduced on palladium black at 50C
for 5 hours. The catalyst is removed by filtration and the filtrate is concentrated under reduced pressure.
A small amount of ethanol is added to the residue and ether is further added to triturate the residue. This procedure is repeated.
Yield: 2.90 g In a small amount of dimethylformamide is dissolved 2.6~ g (0.0023 M) of the compound prepared above, and under ice-cooling a solution of 437 mg (0.0023 M) of p-toluenesulfonic acid in dimethylformamide is added dropwise. After the mlxture is left standing for a whileg the solvent is distilled off in vacuo and ether ` ' ' '' , ~'"~ ''.. '.' .' ~ , ;
~074300 is added to trlturate the residue; Re~ulting powder is dissolved in 20 mQ of dimethylformamide, followed by addition of 823 mg (0.0046 M) of HONB, 0.29 mQ
(0.0023 M) of N-ethylmorpholine and 2.54 g (0.0023 M) PMB BOC
of BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-OH. At -10C, 949 mg (0.0046 M) of dicyclohexylc~rboaiimide in dimethylformamide is added and the mixture is stirred for 3 hours and for further 72 hours at room temperature.
Resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo. Water is added to the residue, and resulting precipitate is collected by filtration and reprecipitated twice from methanol.
Yield: 3.8 g (75.1%) mOp.: 224.0-226.0C (decomp.) [a]25-18.3 (c=0.70, dimethylformamide) Elemental analysis:
Calcd. for Clo9Hl54o26Nl8s2 - C, 59.62; H, 7.069 N, 11.479 S, 2.92 Found Cg 59.559 Hg 6.759 N, 11.32; S, 2.95 (20) Synthesis of H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (somatostatin) In 20 mQ of à mixture of acetic acid and water (8:2 by volume) is dissolved 220 mg (0.1 mM) of PMB BOC BOC
BOC-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr Phe-PMB
Thr-Ser-Cys-OtBuO To the solution is added 171 mg ~0.4 mM) of Hg (CF3C00)2 and the mixture is stirred at - 35 ~
10`~4~(~0 room temperature for 12 hours. Hydrogen sulfide gas is bubbled into the reaction mixture and resulting black precipitate is removed by filtration with celite. The filtrate is concentrated under reduced pressure and the residue is triturated with ether containing a minor amount of ~-mercaptoethanol. Resulting powder is collected by filtration and dried. Yield: 193 mg.
The compound is dissolved in 500 mQ of dimethyl-formamide, followed by addition of 0.28 mQ of 10%
triethylamine-dimethylformamide and further addition of 30 mg of 1,2-diiodoethane. The reaction mixture is stirred at room temperature for 30 minutes and the solvent is distilled off in vacuo. The residue is trifurated with ether and resulting powder is collected by filtration and dried.
Yield: 15~ mg The compound is dissolved in 3 mQ of a mixture of trifluoroacetic acid and water (9:1 by volume) and the solution is shaken at room temperature for 30 minutes. The trifluoroacetic acid is distilled off below 30C under reduced pressure for 10 minutes.
Ether is added3 and resulting precipitate is collected by filtration and dissolved in a small amount of water.
The solution is passed through a column (2.0 x 7.0 cm) of Amberlite IRA-410 tacetate form) to effect ion exchange. The effluent of 60 m~ is passed through a - ~ , 10743~0 column (5.5 x 35.0 cm) of Sephadex LH-20, which is eluted with 0.1 N acetic acid solution. Major fractions of 560-640 mQa~e collected and lyophilized.
Yield: 20.4 mg [~]D6-31.2 (c=0.13, 1% acetic acid) Thin layer chromatography: Rf=0.60 (n-butanol:
pyridine: acetic acid: water=30 : 20 : 6 : 24 by volume, Avicel), Rf=0.40 (n-butanol: ethyl acetate:
acetic acid: water=l : 1 : 1 : 1 by volume, silica gel) Amino acid analysis (6N HCQ , 110C, 24 hours) Lys, 1.97(2); Asp, 1..01(1); Thr, 1.95(2);
Ser, 0.92(1); Gly, 1.00(1); Ala, 1.02(1);
Half Cys, 1.58(2); Phe, 3.01(3)~ average recovery ~2%.
Claims (8)
1. A method for removal of protective group(s) from an amino acid or peptide having thiol group(s) protected with p-methoxybenzyl, 1-adamantyl or t-butyl group, characterized in that the protected amino acid or peptide is treated with mercuric salt of a carboxylic acid of the formula CX3COOH wherein X is hydrogen, chlorine or fluorine.
2. A method as claimed in Claim 1, wherein the carboxylic acid is acetic acid.
3. A method as claimed in Claim 1, wherein the carboxylic acid is trifluoroacetic acid.
4. A method as claimed in Claim 1, wherein the carboxylic acid is trichloroacetic acid.
5. A method as claimed in Claim 1, wherein the amino acid is cysteine.
6. A method as claimed in Claim 1, wherein the peptide has residue(s) of cysteine.
7. A method as claimed in Claim 1, wherein the removal is carried out in the presence of mercuric salt of acetic acid in trifluoroacetic acid.
8. A method as claimed in Claim 1, wherein the removal is conducted in the presence of mercuric salt of trifluoroacetic acid in acetic acid.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP51076676A JPS5848524B2 (en) | 1976-06-28 | 1976-06-28 | Method for removing thiol protecting group |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1074300A true CA1074300A (en) | 1980-03-25 |
Family
ID=13612020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA279,924A Expired CA1074300A (en) | 1976-06-28 | 1977-06-06 | Method for removal of thiol-protecting groups |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5848524B2 (en) |
| CA (1) | CA1074300A (en) |
| FR (1) | FR2361347A1 (en) |
| GB (1) | GB1548101A (en) |
| HU (1) | HU178756B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2451915A1 (en) * | 1979-03-23 | 1980-10-17 | Clin Midy | NEW PROCESS FOR THE PREPARATION OF SOMATOSTATIN |
| JPS62227053A (en) * | 1986-03-28 | 1987-10-06 | Sumitomo Special Metals Co Ltd | High permeability magnetic alloy excellent in workability |
| JPS62227054A (en) * | 1986-03-28 | 1987-10-06 | Sumitomo Special Metals Co Ltd | High permeability magnetic alloy excellent in workability |
| JPH02111838A (en) * | 1988-10-21 | 1990-04-24 | Nippon Steel Corp | Fe-ni magnetic alloy excellent in hot workability and magnetic property |
| JPH04214832A (en) * | 1990-07-30 | 1992-08-05 | Nippon Yakin Kogyo Co Ltd | Fe high-ni alloy excellent in surface property |
-
1976
- 1976-06-28 JP JP51076676A patent/JPS5848524B2/en not_active Expired
-
1977
- 1977-06-06 CA CA279,924A patent/CA1074300A/en not_active Expired
- 1977-06-27 GB GB26762/77A patent/GB1548101A/en not_active Expired
- 1977-06-27 FR FR7719632A patent/FR2361347A1/en active Granted
- 1977-06-27 HU HU77TA1448A patent/HU178756B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| HU178756B (en) | 1982-06-28 |
| FR2361347B1 (en) | 1980-10-17 |
| JPS532404A (en) | 1978-01-11 |
| GB1548101A (en) | 1979-07-04 |
| FR2361347A1 (en) | 1978-03-10 |
| JPS5848524B2 (en) | 1983-10-28 |
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