GB1592969A - Hentriacontapeptides and preparation thereof - Google Patents

Description

(54) HENTRIACONTAPEPTIDES AND PREPARATION THEREOF (71) We, ARMOUR PHARMACEUTICAL COMPANY, (a Delaware corporation), of Greyhound Tower, Phoenix, Arizona 85077, United States of America; do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to hentriacontapeptides and to the preparation thereof; more particularly, it relates to peptides having biological activity and to peptides which may be converted to such biologically active substances. The present invention is particularly concerned with substances having calcitonin-like biological activity or with substances which may be converted to substances having such calcitonin-like activity and with the preparation thereof.

All known natural calcitonin peptides are similar in structure and all contain an amino acid sequence of 32 amino acids. Salmon calcitonin, for example, has the following formula:

Val Leu-Gly-Lys-Leu-Ser-Gln-Glu- 8 9 10 11 12 13 14 15 LeUHis-Lys-Leu-GIn-rhr-Tyrpr0 16 17 18 19 20 21 22 23 ArgThrAsnThrG I ySerGlyThr 24 25 26 27 28 29 30 31 Pro-NH2 32 In U.S. Patent No. 3,926,938 there is disclosed the synthesis of the salmon calcitonin above referred to and in U.S. Patent No. 3,929,758 improved processes by which peptides of this character may easily be prepared are disclosed.

A synthetic peptide having 31 amino acids that has calcitonin-like biological activity has been discovered. Structurally, this peptide is similar to the natural calcitonins, particularly to salmon calcitonin. One significant difference in structure is that, in these synthetic peptides, the amino acid sequence does not contain the tyrosine residue at position 22 of the salmon calcitonin amino acid sequence. Chemically, the synthetic peptide is more stable because it does not contain the oxidatively-labile tyrosine residue. This peptide is also more economically produced since it contains one less amino acid. The synthetic peptide has a biological activity similar in potency and quality to salmon calcitonin.

A solid phase type of synthesis is used. A starting material is a resin called benzhydryl amine resin (BHA resin). This resin is derived from a cross-linked polystyrene bead resin obtained by copolymerization of styrene and divinylbenzene. Resin of this type is known and its preparation is further demonstrated by Pietta et al (Pietta, P. S. and Marshall, G.

R., Chem. Commun., 650 [1970]). This cross-linked polystyrene BHA resin is available commercially. The designation:

is used herein to represent the BHA resin wherein (g) represents the polystyrene portion of the resin.

Resin peptide synthesis In this synthesis, the amino acids are added one at a time to the insoluble resin until the total peptide sequence has been built up on the resin. The functional groups of the amino acids are protected by blocking groups. The a-amino group of the amino acids is protected by a tertiary butyloxycarbonyl group. This a-tertiary butyloxycarbonyl group is designated as "BOC". The hydroxyl functions of serine and threonine are protected by beazyl, 4-methoxybenzyl, 4-methylbenzyl, 3, 4-dimethylbenzyl, 4-chlorobenzyl, 2,6dichlorobenzyl, 4-nitrobenzyl or benzhydryl. The symbol "BZ" is used to represent such a group.

The thiol function of cysteine is protected by such a BZ group or by an n-alkylthio group, such as methylthio, ethylthio, n-propylthio or n-butylthio. Herein, the symbol R7 is used to represent an n-alkylthio group or BZ and the symbol R1 to represent BZ when R7 represents n-alkylthio and to represent n-alkylthio when R7 represents BZ. The guanidino function of arginine is protected by a nitro group or a tosyl group. The character T is used herein to represent a nitro group or a tosyl group. The E-amino function of lysine is protected by benzyloxycarbonyl, 2-chlorobenzyl-oxycarbonyl, 2-bromobenzyloxycarbonyl or 3,4-dimethyl-benzyloxycarbonyl. The character V is used to represent such a group. The protective groups V may also be used on the imidazole nitrogen of histidine. The above-mentioned protective group BZ may also be used to protect the y-carboxylic acid group of glutamic acid.

In its broadest aspect, the present invention relates to a peptide containing the following structure: Thr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 and to the corresponding protected resin peptide, as well as to the preparation thereof.

The sequence of the present synthetic peptide having calcitonin-like activity may be presented as follows:

Val -Leu-Gly-Lys-Leu-Ser-Gln-Clu- 8 9 10 11 12 13 14 15 Leu--H 15 --Lyss-Leeu-Gln --Thrr-Pro --Arg - 16 17 18 19 20 21 22 23 Thr-Asn -Thr - Gly - Ser - Gly - Thr - 24 25 26 27 28 29 30 Pro-NH2 31 As may be seen from the above sequence, 31 amino acids are present, the positions being numbered according to the accepted custom beginning at position 1 for the CYS on one end of the chain and ending with PRO at position 31 at the other end of the chain. (For clarity, this same numbering system will be followed in referring to the cycles of the synthesis. The assembly of the amino acids begins with cycle 31 which involves the coupling of proline and continues with cycle 30 which involves the coupling of threonine.) etc.

Preferred amino acid reactants for use in each of the 31 cycles of the synthesis are given in the following Table: TABLE Cycle Number Amino Acid Reactant 31 BOC-L-proline 30 BOC-O-benzyl-L-threonine 29 BOC-glycine 28 BOC-O-benzyl-L-serine 27 BOC-glycine 26 BOC-O-benzyl-L-threonine 25 BOC-L-asparagine p-nitrophenyl ester 24 BOC-O-benzyl-L-threonine 23 BOC-Q-nitro-L-arginine or BOC-Q-tosyl-L-arginine 22 BOC-L-proline 21 BOC-O-benzyl-L-threonine 20 BOC-L-glutamine-p-nitrophenyl ester 19 BOC-L-leucine 18 BOC-E-CBZ-L-lysine or BOC~E~2~ chlorobenzyloxycarbonyl-L-lysine 17 BOC-N(im)-CBZ-L-histidine 16 BOC-L-leucine 15 BOC-L-glutamic acid y-benzyl ester 14 BOC-L-glutamine p-nitrophenyl ester 13 BOC-O-benzyl-L-serine 12 BOC-L-leucine 11 BOC-E-CBZ-L-lysine or B O C- E-2-chlorobenzyloxycarbonyl-l-lysine 10 BOC-glycine 9 BOC-L-leucine 8 BOC-L-valine 7 BOC-S-ethylthio-L-cysteine, BOC-S-methylthio-L-cysteine, BOC-S-n-propylthio-L-cysteine or BOC-S-n-butylthio-L-cysteine 6 BOC-O-benzyl-L-threonine 5 BOC-O-benzyl-L-serine 4 BOC-L-leucine 3 BOC-L-asparagine p-nitrophenyl ester 2 BOC-O-benzyl-L-serine 1 BOC-S-p-methoxybenzyl-L-cysteine, BOC-S-benzyl-L-cysteine or BOC-S-3 ,4-dimethylbenzyl-L-cysteine Each of the amino acid derivatives mentioned in the Table are available commercially, except, perhaps, the derivative mentioned for use in cycle No. 7. These materials useful in cycle 7 may be prepared according to the method described in the literature (U. Weber and P. Hartter, Hoppe-Seyler's, Z. Physiol. Chem. 351, 1384-8 [1970]).

Cycle 31 Coupling of L-Proline to BHA Resin The reaction vessel used in all steps of the resin peptide synthesis may be a glass vessel equipped with inlet ports at the top for addition of materials and a sintered glass disk at the bottom for removal of soluble reaction mixtures and wash solvents by filtration. Filtration may be performed either by vacuum or the use of nitrogen pressure. The contents of the vessel may be agitated by shaking the entire vessel or by a mechanical stirrer.

In cycle 31, the BHA resin is placed in the reaction vessel and suspended in a solvent, such as methylene chloride, chloroform, dimethylformamide or benzene, in proportions of from 3 to 12 ml of solvent per gram of resin. To this is added BOC-L-proline in an amount of from 1 to 6 equivalents per free amine equivalent of the BHA resin employed. After a period of mixing of from 5 to 10 minutes, a diimide coupling reagent (CA), such as dicyclohexyl carbodiimide (DCC), is added. The diimide coupling agent is generally used in an amount of from 0.5 to 2.0 equivalents per equivalent of BOC-L-proline used.

The BOC-L-proline may be coupled in the absence of a coupling reagent if its active ester derivative, its azide derivative, its symetrical anhydride derivative, or an appropriate mixed anhydride derivative is used. The active ester derivatives that may be employed include 2-nitrophenyl ester, 4-nitrophenyl ester, pentafluorophenyl ester and N-hydroxysuccimide ester. The active esters are generally used in amounts of from 1 to 10 equivalents per free amine equivalent of BHA resin.

The reaction mixture consisting of the BHA resin, the solvent, the BOC-L-proline and the coupling reagent or BOC-L-proline active ester is stirred or shaken mechanically until the reaction is complete as is indicated by a ninhydrin test (E. Kaiser, et al., Anal.

Biochem., 34 595-8 [1970]) on the test sample. After completion of the coupling reaction, the BOC-L-proline resin may be washed with solvents, such a methylene chloride, chloroform, methyl alcohol, benzene, dimethylformamide or acetic acid. The amount of wash solvent may suitably be from 5 to 20 ml of solvent per gram of BHA resin used initially. If it is desired to terminate the coupling reaction before completion, the washing procedure is used and the remaining free amino groups on the BOC-L-proline resin may be blocked from further reaction by acetylation using an excess of acetylation reagents. The acetylation procedure is performed by agitating the BOC-L-proline resin with a solution of the acetylation reagent for a period of from 0.5 to 12 hours. Acetylation reagents, such as N-acetylimidazole in methylene chloride solution or a mixture of acetic anhydride and triethylamine in chloroform may be used. The acetylation reagent may be used in the amount of from 0.5 to 5.0 equivalents per equivalent of free amine titer of the starting BHA resin.

The coupling reaction to produce the BOC-L-proline resin may be illustrated by the following:

Deprotection of BOC-L-proline Resin The BOC-L-proline resin produced as described above may be washed with a solvent, such as those mentioned above, and deprotected by agitating it with an agent, such as a mixture of trifluoroacetic acid (TFA) in a solvent, such as methylene chloride, chloroform or benzene. The amount of TFA in the solvent may vary from 10 to 100% v/v of the mixture. The amount of TFA-solvent mixture may vary from 3 to 20 ml per gram of BHA resin used initially. The reaction time may be from 10 minutes to 4 hours. The deprotection step is terminated by filtration to remove the TFA-solvent mixture. The residual TFA may be removed from the L-proline resin by washing with from 3 to 20 ml per gram of BHA resin of a solution of from 5 to 30% v/v of triethylamine in a solvent, such as methylene chloride, chloroform or benzene. Other tertiary or secondary organic amines may be used in place of the triethylamine, such as trimethylamine, N-ethylpiperidine or diisopropylamine. The free amine titer of the L-proline resin may be determined by the Dorman titration procedure (Dorman, L.C., Tetrahedron Letters, 1969 2319-21). The deprotection reaction may be illustrated by the following:

Cycle 30 The L-prolyl BHA resin obtained as a result of cycle 31 may be suspended in a coupling solvent, the BOC-O-BZ-L-threonine derivative added and the mixture equilibrated in the same manner. The coupling agent, DCC, may be added, and, after completion of the reaction as indicated by the ninhydrin test, the reaction mixture is removed from the BOC-O-BZ-L-threonyl-L-prolyl BHA resin by filtration. The peptide resin may be washed with solvents. The amounts of reactants and solvents and reaction times may be the same as described in cycle 31. The BOC group may be removed from the peptide resin by the deprotection method described in cycle 31. The resulting O-BZ-L-threonyl-L-prolyl BHA resin is then ready for cycle 29. The reactions of cycle 30 may be illustrated by the following:

For convenience, the resulting resin peptide may be represented, using abbreviated nomenclature, as follows:

Cycle 29 In cycle 29, the coupling reaction and also the deprotection reaction may be performed in the same manner as in cycle 30, except that BOC-glycine is used in place of BOC-O-BZ-L-threonine. The reaction through coupling and deprotection may be represented as follows:

Cycle 28 In cycle 28, the coupling and deprotection reactions may be performed in the same manner as in cycle 30, except for the substitution of BOC-O-BZ-L-serine as the amino acid derivative. This may be represented:

Cycle 27 In cycle 27, the coupling and deprotection reactions are performed as described in cycle 30, except that BOC-glycine is substituted as the amino acid reactant. These reactions through coupling and deprotection may be represented as follows:

Cycle 26 In this cycle, the coupling and deprotection reactions may be as in cycle 30 using the same amino acid reactant, resulting in the following compound:

Cycle 25 In cycle 25, the coupling reaction is performed using an active ester derivative of BOC-L-asparagine. The active ester procedure is used in place of the DCC coupling method to avoid a known side reaction that occurs with the use of DCC coupling agent with BOC-asparagine or BOC-glutamine. The reaction is performed using the active ester derivative of BOC-L-asparagine in an amount of from 2 to 10 equivalents per free amine equivalent of BHA resin in, for example, dimethylformamide, mixtures of dimethylformamide with benzene, methylene chloride or chloroform in amounts of from 2 to 20 ml. of solvent per gram of BHA resin used initially. Reaction times are from 1 to 72 hours. The reaction mixture is removed from the BOC-peptide resin by filtration after completion of the reaction as indicated by a ninhydrin test. The active esters derivative employed may be 2-nitrophenyl esters, 4-nitrophenyl esters or pentafluorophenyl. The symbol AE is used to designate the active ester portion of the derivative. The coupling reaction may be represented:

The deprotection reaction to remove the BOC group is performed as in cycle 31.

Cycles 24 to 21 In each of cycles 24 to 21, the coupling and de-protection reactions may be conducted using methods and proportions of reactants similar to those used in cycle 30 using BOC-BZ-L-threonine in cycle 24, BOC-o T-L-arginine in cycle 23, BOC-L-proline in cycle 22, and BOC-O-BZ-L-threonine in cycle 21. The compound resulting from the completion of cycle 21 may be represented as follows:

This compound is considered to be novel and, as will be shown below, may be converted to a peptide having calcitonin-like activity.

In one embodiment, the present invention relates to a process for the preparation of the above-mentioned resin peptide which comprises mixing

with-BOC-Thr(Bz) - OH or an active ester, an azide or an anhydride thereof and, unless the active ester, azide or anhydride is used, in the presence of a diimide followed by deprotection using trifluoroacetic acid.

Cycle 20 In cycle 20, the coupling and deprotection reactions may be performed using the methods and proportions of reactants as in cycle 25 using a BOC-L-glutamine active ester derivative as the amino acid derivative, resulting in the compound:

Cycle 19 In cycle 19, the reactions are performed as in cycle 31 using BOC-L-leucine as the amino acid derivative. The compound resulting from cycle 19 is:

Cycle 18 In cycle 18, the amino acid derivative BOC-E-V-L-lysine may be used. Otherwise, cycle 18 may be performed as in cycle 30 resulting in the compound:

Cycles 17 to 15 Cycles 17 to 15 may be performed as in cycle 31, except for the use of BOC-N(im)-V-L histidine in cycle 17, BOC-L-leucine as the reactant in cycle 16 and BOC-L-glutamic acid BZ ester as the reactant in cycle 15, resulting in the following compound from cycle 15:

Cycles 14 to 8 Cycle 14 may be performed identically to cycle 20 using BOC-L-glutamine-AE as the amino acid derivative. Cycles 13 to 8 may be performed as in cycle 31, except for the use of BOC-O-BZ-L-serine in cycle 13, BOC-L-leucine in cycle 12, BOC-E-V-L-lysine in cycle 11, BOC-glycine in cycle 10, BOC-L-leucine in cycle 9 and BOC-L-valine in cycle 8 resulting in the compound:

Cycle 7 Cycle 7 may be performed as in cycle 31, except for the use of BOC-S-R-L-cysteine for the amino acid derivative. The compounds resulting from cycle 7 are represented as follows:

wherein R7 represents n-alkylthio.

Cycles 6 to 2 Cycles 6 to 4 were performed as in cycle 31, except that BOC-O-BZ-L-threonine was used as the amino acid derivative in cycle 6, BOC-BZ-L-serine was used as the amino acid derivative in cycle 5 and BOC-L-leucine was used in cycle 4 as the amino acid derivative.

Cycle 3 may be performed identically to cycle 26 using BOC-L-asparagine active ester. In cycle 2, the procedures may be the same as cycle 28 using BOC-O-BZ-L-serine as the amino acid derivative. The compound resulting from cycle 2 may be represented as follows:

Cycle I This cycle may be performed identically to cycle 7 using BOC-S-R1-L-cysteine derivatives. The Rl group chosen for the cysteine may be the same as that used in cycle 7 or different. For example, if the derivative chosen for cycle 7 is BOC-S-ethylthio-L-cysteine, the derivative in cycle 1 may be BOC-S-4-methoxybenzyl-L-cysteine or if BOC-S-4 methoxybenzyl-L-cysteine was chosen for cycle 7, then the other derivative may be used in cycle 1. The compounds resulting from cycle 1 are represented as follows:

wherein one of R, and R7 'represents n-alkylthio and the other represents BZ.

Cycle 1 represents the completion of the resin peptide. The resin peptide may be removed from the reaction vessel and dried in a vacuum. The weight of the resin peptide may be expected to be from 2.0 to 3.5 times the weight of BHA resin used initially in the synthesis.

Resin Pep tide Cleavage The peptide is cleaved from the resin peptide resulting from cycle 1 by treatment with liquid hydrogen fluoride (HF). The HF cleavage reaction may be performed by treating a mixture of the resin peptide and anisole (from 0.5 to 5 ml per gram of resin peptide) with liquid HF (from 2 to 20 ml per gram of resin peptide) for from 0.5 to 20 hours at from 20 to + 150C. After the reaction period, the excess HF may be removed by evaporation and the resulting mixture of peptide and rasin beads may be extracted with an organic solvent, such as ethyl acetate, diethyl ether or benzene, to remove the anisole and residual HF. The peptide may be separated from the resin beads by extraction into aqueous acetic acid. The peptide at this stage is not cyclic, but is the non-cyclic product without the cyclic disulfide bond between the cysteine residues at positions 1 and 7 in the molecule.

The HF treatment removes all blocking groups from the peptide, except the S-alkylthio blocking groups on the thiol function of the cysteine residue at position 7 or position 1. The S-n-alkylthio-L-cysteine residue is stable to the HF cleavage procedure and remains intact throughout the cleavage and extraction procedures. The S-BZ-L-cysteine residue is cleaved by HF to yield a cysteine residue having a free thiol function. Both types of blocking groups have been employed during the synthesis in combination with each other at positions 7 and 1. Thus, the peptides obtained after HF cleavage may be one of two types depending upon the blocking groups chosen for the thiol function of the cysteine derivative used during the resin peptide synthesis.

If BOC-S-Z-L-cysteine derivatives were used in the resin peptide synthesis cycle 1 and BOC-S-ethylthio-L-cysteine was used in cycle 7, the peptide resulting after HF cleavage would be of Type 1 and would have a free thiol function at postion 1 and have a S-ethylthio function on the cysteine residue at position 7. The peptide would be represented as follows: Type I

S-n-alkyl H-CYS-SER-ASN-LEU-SER-THR-CYS-VAL-LEU-GLU LYS-LEU-SER-GLN- GLU-LEU-HIS-LYS--LEU- GLN-THR- PRO-ARG-THR-ASN-THR-GLY-SER-OLY-THR-PRO-NH2 Conversely, if BOC-S-n-alkylthio-L-cysteine derivative was used in cycle 1 and BOC-S-BZ-L-cysteine was used in position 7, the peptide resulting from the cleavage would be of "Type II" and would be represented as follows: Type 11

S-n-alkyl H-CYS-SER-ASN-LEU-SER-THR-CYS-VAL-LEU-GLY LYS-LIEU-SER- GLN- GLU-LEU-HIS-LYS-LEU-GLN-THR-PRO- ARG-THR-ASN-THR-GLY-SER-GLY-THR-PRO-NH2 For example the conversion of Type I and II peptides to the cyclic disulphide peptide may be performed by diluting with distilled water the aqueous acetic acid solution of the crude peptides from HF cleavage to a final volume of from 50 to 200 ml per gram of resin peptide cleaved. The pH of this solution is adjusted to from 5 to 10 by the addition of ammonium hydroxide solution and the mixture is stirred in a closed container under a stream of an inert gas, such as nitrogen, for from 2 to 48 hours. The reaction may be stopped when the off-gas stream no longer containes n-alkylmercaptan. The pH of the reaction mixture may be lowered to from 3.5 to 5.5 by the addition of glacial acetic acid.

In one embodiment, the present invention relates to a process for the preparation of a peptide having a cysteine disulphide linkage between positions 1 and 7 which comprises holding a peptide having either a Type I or a Type II structure in a solution substantially free of oxygen.

The solutions may be an aqueous alcoholic solution.

The peptide has now been converted to a peptide having calcitonin-like biological properties. The crude peptide solution obtained may be purified chromatographically to yield a freeze-dried product similar in chemical properties and biological activity to natural salmon calcitonin.

Purification of the crude synthetic calcitonin The crude peptide solutions at pH 5.0 from the above synthesis may be concentrated using an ion-exchange procedure. The concentrate may be purified by a combination of gel-filtration procedures and ion-exchange chromatography methods. The final purified product may be obtained from solution by freeze-drying as a fluffy white solid. The product gives the correct amino acid analysis for the desired peptide.

Following is a specific example of the preparation of the peptide.

EXAMPLE 1 Resin activation The BHA resin (5 g) with an amine titer of 0.61 meq/g was placed in the reactor vessel of a peptide synthesizer marketed by Schwarz-Mann, Inc. of Orangeburg, New York, U.S.A.

The resin was treated with 25 ml of the following solvents filtering after each treatment: Methylene chloride for 2 minutes Chloroform for 2 minutes two times each 10% v/v triethylamine in chloroform for 5 minutes two times each Chloroform for 2 minutes Methylene chloroide for 2 minutes three times each Cycle 31 Coupling The BHA resin, 25 ml. of methylene chloride and 1.31 g. (0.0061 moles) of BOC-L-proline was stirred for 10 minutes. 6.1 ml. of a methylene chloride solution of dicyclohexylcarbodiimide (1 milliequivalent of DCCI per 1 ml. of solution) was added to the reactor and the mixture agitated for 6 hours. The reaction mixture was removed from the reactor by filtration and the BOC-L-prolyl BHA resin subjected to the following successive 2 minute, 25 ml. washes, removing the wash by filtration each time: Methylene chloride two times Methyl alcohol two times Methylene chloride three times Acetylation The resin was then agitated with a mixture of 1.5 ml. of triethylamine (TEA), 1 ml. of acetic anhydride and 25 ml. of chloroform for 2 hours. The reaction mixture was removed by filtration and the resin subjected to the following 2 minute, 25 ml. washes: Chloroform two times Methyl alochol two times Methylene chloride three times Deprotection The BOC-protected resin was agitated for 5 minutes with a mixture of 15 ml. of trifluoroacetic acid (TFA) and 15 ml. of methylene chloride. This mixture was removed by filtration and the resin was agitated with a second mixture of 15 ml. of TFA and 15 ml. of methylene chloride for 30 minutes. The reaction mixture was removed by filtration and the resin subjected to the following 25 ml. washes: Methylene chloride two times two minutes each Methyl alcohol two times two minutes each Chloroform two times two minutes each 10% v/v TEA in chloroform two times ten minutes each Methylene chloride two times two minutes each The L-proline BHA resin was titrated to establish the amine or proline titer. This value was 0.55 milliequivalents of amine or proline per gram of resin.

Cycle 30 Coupling The L-prolyl resin, 25 ml of methylene chloride and 1.64 g (0.0053 mole) of BOC-O-benzyl-L-threonine were agitated for 10 minutes. Then, 5.5 ml of a methylene chloride solution of dicyclohexylcarbodiimide (1 milliequivalent of DCCI per 1 ml of solution or a total of 0.0055 mole of DCCI) was ,added to the reactor and the mixture agitated for 2 hours. The reaction mixture was removed from the reactor and the resin was subjected to the following successive 2 minute, 25 ml washes, removing the wash by filtration each time.

Methylene chloride two times Methyl alcohol two times Methylene chloride three times.

A ninhydrin test was negative.

Deprotection The deprotection procedure described in cycle 31 was repeated for this cycle.

Cycles 29 to 26 The coupling and deprotection procedures used in these cycles were the same as in cycle 30, except that the following amino acid derivatives were used in place of the threonine derivative: Cycle 29 -- 0.93 g (5).0053 mole) of BOC glycine Cycle 28 -- 1.55 g (0.0053 mole) of BOC-O-benzyl-L-serine Cycle 27 -- The reactant used was the same as cycle 29 Cycle 26 -- The reactant used was the same as cycle 30.

Cycle 25 Coupling The peptide resin obtained from cycle 26 was washed twice with 25 ml portions of dimethylformamide (DMF). The resin was then agitated for 24 hours with a solution of 2.82 g (0.008 mole) of BOC-L-asparagine p-nitrophenyl ester in 35 ml of DMF. The reaction mixture was filtered and the resin peptide subjected to two minute washes with two successive 25 ml portions of the following solvents: DMF, methylene chloride, methanol, methylene chloride. Each individual solvent was removed by filteration. A ninhydrin test was negative.

Deprotection The deprotection procedure used in cycle 31 was repeated.

Cycle 24 Coupling and deprotection procedures were the same as in cycle 30 using the same reactants and amounts.

Cycle 23 Coupling The resin peptide obtained from cycle 25 was washed with two successive 25 ml.

portions of DMF. The resin peptide was then agitated for 10 minutes with a mixture of 3.42 g (0.008 mole) of BOC-N-y-tosyl-L-arginine and 25 ml of DMF. Then, 8 ml of DCCI in methylene chloride (equivalent to 0.008 mole of DCCI) was added and the mixture agitated for 6 hours. The reaction mixture was removed by filtration. The resin peptide was subjected to two minute washes with two successive 25 ml portions of the following solvents: DMF, methylene chloride methyl alcohol, methylene chloride. The ninhydrin test was negative.

Deprotection Repeat deprotection procedures used in cycle 31.

Cycle 22 Coupling The peptide resin obtained from cycle 23 was agitated for 10 minutes with 1.72 g (0.008 mole) of BOC-L-proline and 25 ml of methylene chloride. 8 ml. of DCCI in methylene chloride (equivalent to 0.008 mole of DCCI) was added and the mixture agitated for 6 hours. The reaction mixture was removed by filtration and the resin peptide subjected to two minute washes with two successive 25 ml portions of the following solvents: methylene chloride, methyl alcohol, methylene chloride. Each individual wash was removed by filtration. The ninhydrin test was negative.

Deprotection The deprotection procedure used in cycle 31 was repeated.

Cycle 21 The coupling and deprotection procedures used in these cycles were the same as in cycle 22, except that in the coupling reaction the following amino acid derivative was used in place of BOC-L-proline.

Cycle 21 -- 2.47 g (0.008 mole) of BOC-O-benzyl-L-threonine Cycle 20 This procedure is the same as cycle 25, except that 2.94 g (0.008 mole) of BOC-L-glutamine p-nitrophenyl ester is used in place of the asparagine derivative.

Cycles 19 to 15 The procedure is the same as used in cycle 30, except that the following amino acid derivatives were used in place of the threonine derivative: Cycle 19 -- 1.32 g (0.0053 mole) of BOC-L-leucine Cycle 18 -- 2.20 g (0.0053 mole) of BOC-E-2-chlorocarbobenzyloxy-L-lysine Cycle 17 -- 2.06 g (0.0053 mole) of BOC-N(im)-carbobenzyloxy-L-histidine Cycle 16 -- See cycle

Cycle 1 The procedures used were the same as used in cycle 30, except that 1.81 g (0.0053 mole) of BOC-S-p-methoxybenzyl-L-cysteine was used in place of the threonine derivative.

After completion of cycle 1, the resin peptide was washed with two successive 25 ml.

portions of n-hexane. The peptide material was removed from the reactor and dried in an electric vacuum oven at 400C. and 0.1 mm of Hg for 24 hours.

Cleavage with hydrogen fluoride The dried resin peptide (10 g) and 10 ml of anisole were placed in a Teflon (Registered Trade Mark) reaction vessel. The vessel equipped with a Teflon-coated magnetic stirrer was placed in a dry ice-acetone bath and 75 ml. of hydrogen fluoride gas was condensed into the vessel. This mixture was stirred at OOC in an ice bath for 1 hour. The hydrogen fluoride was removed by evaporation at reduced pressure. The residue was triturated with six 75 ml portions of ethyl acetate. The peptide was extracted from the resin beads with 600 ml of 0.1 molar aqueous acetic solution.

Cyclization of the pep tide The aqueous acetic acid extract obtained from hydrogen fluoride cleavage was diluted to 1.2 litres by addition of 700 ml of distilled water. The pH of the solution was adjusted to 7.5 by the addition of concentrated ammonium hydroxide. The solution was stirred in a closed vessel under a stream of nitrogen for 24 hours. At this time no ethyl mercaptan'could be detected in the emerging nitrogen stream. The ethyl mercaptain content of the nitrogen stream was measured by passing the stream through a solution of Ellman's reagent (Ellman, G.L., Arch. Biochem. Biophys., 82, 70-7 (1959). The pH of the reaction mixture was adjusted to 5.0 by addition of glacial acetic acid.

Purification of the crude hentriacontapeptide The 1.2 litres of solution from the above synthesis at pH 5.0 was concentrated using a SP-Sephadex (Registered Trade Mark) C-25 ion-exchange column. The 75 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted and purified by passing through a Sephadex G-25 (fine) gel-filtration column and eluting with 0.03 molar aqueous acetic acid solution. The hentriacontapeptide fraction from this column was adjusted to pH 6.0 by addition of ammonium hydroxide solution. This solution was further purified by ion-exchange chromatography using a Whatman (Registered Trade Mark) CM52 column eluted with ammonium acetate buffer. The hentriacontapeptide fraction from this column was adjusted to pH 5.0 by addition of glacial acetic acid. This solution was concentrated using a SP-Sephadex C-25 ion-exchange column. The 30 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted using a Sephadex G-25 (fine) gel-filtration column. The purified peptide fraction was collected and freeze-dried. The product was obtained as a fluffy white solid. Amine acid analysis of the product gave the following ratios with the theoretical values given in parenthesis: Asp 1.9 (2), Thr 5.4 (5), Ser 4.1 (4), Glu 2.9 (3), Pro 2.1 (2), Gly 3.0 (3), Val 0.8 (1), Leu 5.0 (5), His 0.9 (1), Lys 1.9 (2), Arg 0.9 (1). This product assayed at 6000 MRC units per mg.

WHAT WE CLAIM IS: 1. A resin peptide containing the following structure:

wherein, Bz represents benzyl, 4-methoxybenzyl, 4-methylbenzyl, 3,4-dimethylbenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl or benzhydryl; T represents nitro or tosyl; and (ÉD represents polystyrene cross-linked by divinylbenzene.

2. A resin peptide as claimed in claim 1 having the following structure:

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (15)

**WARNING** start of CLMS field may overlap end of DESC **. Cycle 1 The procedures used were the same as used in cycle 30, except that 1.81 g (0.0053 mole) of BOC-S-p-methoxybenzyl-L-cysteine was used in place of the threonine derivative. After completion of cycle 1, the resin peptide was washed with two successive 25 ml. portions of n-hexane. The peptide material was removed from the reactor and dried in an electric vacuum oven at 400C. and 0.1 mm of Hg for 24 hours. Cleavage with hydrogen fluoride The dried resin peptide (10 g) and 10 ml of anisole were placed in a Teflon (Registered Trade Mark) reaction vessel. The vessel equipped with a Teflon-coated magnetic stirrer was placed in a dry ice-acetone bath and 75 ml. of hydrogen fluoride gas was condensed into the vessel. This mixture was stirred at OOC in an ice bath for 1 hour. The hydrogen fluoride was removed by evaporation at reduced pressure. The residue was triturated with six 75 ml portions of ethyl acetate. The peptide was extracted from the resin beads with 600 ml of 0.1 molar aqueous acetic solution. Cyclization of the pep tide The aqueous acetic acid extract obtained from hydrogen fluoride cleavage was diluted to 1.2 litres by addition of 700 ml of distilled water. The pH of the solution was adjusted to 7.5 by the addition of concentrated ammonium hydroxide. The solution was stirred in a closed vessel under a stream of nitrogen for 24 hours. At this time no ethyl mercaptan'could be detected in the emerging nitrogen stream. The ethyl mercaptain content of the nitrogen stream was measured by passing the stream through a solution of Ellman's reagent (Ellman, G.L., Arch. Biochem. Biophys., 82, 70-7 (1959). The pH of the reaction mixture was adjusted to 5.0 by addition of glacial acetic acid. Purification of the crude hentriacontapeptide The 1.2 litres of solution from the above synthesis at pH 5.0 was concentrated using a SP-Sephadex (Registered Trade Mark) C-25 ion-exchange column. The 75 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted and purified by passing through a Sephadex G-25 (fine) gel-filtration column and eluting with 0.03 molar aqueous acetic acid solution. The hentriacontapeptide fraction from this column was adjusted to pH 6.0 by addition of ammonium hydroxide solution. This solution was further purified by ion-exchange chromatography using a Whatman (Registered Trade Mark) CM52 column eluted with ammonium acetate buffer. The hentriacontapeptide fraction from this column was adjusted to pH 5.0 by addition of glacial acetic acid. This solution was concentrated using a SP-Sephadex C-25 ion-exchange column. The 30 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted using a Sephadex G-25 (fine) gel-filtration column. The purified peptide fraction was collected and freeze-dried. The product was obtained as a fluffy white solid. Amine acid analysis of the product gave the following ratios with the theoretical values given in parenthesis: Asp 1.9 (2), Thr 5.4 (5), Ser 4.1 (4), Glu 2.9 (3), Pro 2.1 (2), Gly 3.0 (3), Val 0.8 (1), Leu 5.0 (5), His 0.9 (1), Lys 1.9 (2), Arg 0.9 (1). This product assayed at 6000 MRC units per mg. WHAT WE CLAIM IS:

1. A resin peptide containing the following structure:

wherein, Bz represents benzyl, 4-methoxybenzyl, 4-methylbenzyl, 3,4-dimethylbenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl or benzhydryl; T represents nitro or tosyl; and (ÉD represents polystyrene cross-linked by divinylbenzene.

2. A resin peptide as claimed in claim 1 having the following structure:

wherein, Bz, T and Q are as defined in claim 1.

3. A resin peptide as claimed in claim 1 having the following structure:

wherein, Bz, T and R are as defined as claim 1; V represents benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 2chlorobenzyloxycarbonyl or 3,4-dimethylbenzyloxycarbonyl, and one of R1 and R represents n-alkylthio and the the other of R1 and R7 represents Bz.

4. A peptide containing the following structure: -Thr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Oly-Thr-Prn-NH2

5. A peptide as claimed in claim 4 having the following structure:

Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Pro-Arg-Thr-Asn-Thr- Oly-Ser-Oly-Thr-Pro-NH2 wherein one of Rl and R7 represents n-alkylthio and the other of Ri and R7 represents hydrogen.

6. A peptide having the following structure:

7. A resin peptide or peptide as claimed in any of claims 1 to 6 substantially as herein described.

8. A process for the preparation of a resin peptide as claimed in claim 1 which comprises mixing a resin peptide having the following structure:

with BOC-Thr(Bz)-OH or an active ester, an azide or an anhydride thereof and, unless the active ester, azide or anhydride is used, in the presence of a diimide followed by deprotection using trifluoracetic acid; wherein Bz, T and Q are as defined in claim 1.

9. A process for the preparation of a resin peptide as claimed in claim 2 which comprises mixing

with BOC-Thr(Bz)-OH or an active ester, an azide or an anhydride thereof and, unless the active ester, azide or anhydride is used, in the presence of the diimide followed by deprotection using trifluoracetic acid to obtain

wherein, Bz, T and Q are as defined in claim 1.

10. A process for the preparation of a peptide as claimed in claim 4 which comprises deprotecting a resin peptide as claimed in claim 1 using hydrogen fluoride.

11. A process for preparing a peptide as claimed in claim 6 having a cysteine disulphide linkage between positions 1 and 7 which comprises holding a peptide having the following structure:

LeuHisLys-Leu-Oln-Thr-Pro-Arg-Thr-Asn-Thr-Oly-Ser- Gly-Thr-Pro-NH2 wherein, R7 represents n-alkylthio or hydrogen; and R1 represents n-alkylthio when R7 represents hydrogen or hydrogen when R represents n-alkylthio, in a solution substantially free of oxygen.

12. A process as claimed in claim 11 in which the peptide is held in the solution at a pH of from 5.0 to 10.0 adjusted by ammonium hydroxide solution.

13. A process as claimed in claim 11 or claim 12 in which the solution is an aqueous alcoholic solution.

14. A process as claimed in any of claims 8 to 13 substantially as herein described.

15. A resin peptide or peptide as claimed in claim 4 when prepared by a process as claimed in any of claims 8 to 14.