DE2649727A1 - CARRIED PEPTIDES, PTH-PEPTIDE INTERMEDIATES AND THEIR MANUFACTURING - Google Patents

Description

The invention relates to peptide synthesis, in particular to carrier-bound peptides which are used for the production biologically active peptides can be used. The invention encompasses both the peptides and new compounds as well as their manufacturing process.

For a long time, certain natural biologically active substances have been obtained from the material of animal glands, which are used in human medicine for the treatment of diseases and deficiency symptoms. One such substance is the parathyroid hormone, commonly referred to as PTH for short, which is long-lasting

542- (case 5972) -FSBk

70981871082

animal parathyroid glands, especially pigs and cattle.

The great effort associated with the recovery of the relatively small parathyroid glands from animals immediately after slaughter, the limited amount of glands obtained in this way and the complex purification process required for the production of peptides that can be used in human medicine are extremely large disadvantages Production of natural peptide hormones from animal glands. Although there is consequently an extraordinary interest in the development of practical methods as well as new compounds that enable the commercial synthesis of peptides such as human parathyroid hormone (HPTH), to the knowledge of the inventors, none has been so far procedural or material approach found to solve this problem.

For the human Nebenschild'drüsenhormon (HPTH) a sequence of 84 amino acids was determined, the amino terminal sequence \ - ~ 5K having the following structure:

Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

The abbreviations Phe, Asn, His etc. stand for the different amino acid groups in the peptide chain; the numbers represent the positions of the amino acid group in the chain according to the usual nomenclature (cf. Niall et al., Proc. Nat. Acad. Sci. USA, 71 (1974)

709818/1082

384-83). The above fragment obviously has the full biological value in comparison with the entire molecule Activity.

Some laboratory procedures are available for the synthesis of certain peptides of relatively short amino acid chain length has been proposed; to be mentioned in this regard: R.B. Merrifield: "Solid Phase Peptide Synthesis. I." The Synthesis of a Tetrapeptide ", J. Am. Chem. Soc. Vol. 85 (1963), 2149-54; J.V /. Stewart and J.D. Young: "Solid Phase Peptide Synthesis", V / .H. Freeman Company, San Francisco, California. However, no information whatsoever can be found in these publications via carrier-bound peptides with amino acids of the type and sequence according to the invention.

The invention is based on the object of specifying intermediate carrier-bound peptides from which biologically active peptides can be derived, in particular peptides with the activity of human parathyroid hormone, and a method for the commercial production of such peptides. The object is according to the invention dissolved by a synthesis in or on a solid phase, initially an insoluble polystyrene resin, the is obtained by catalytic polymerization of styrene and divinylbenzene, is chloromethylated, whereupon the Resin first phenylalanine, then asparagine and the other amino acids of the chain in a given sequence below Use of a system of formation and deprotection of the active amino and carboxyl groups can be synthesized. After coupling the last amino acid in the chain, the peptide chains are removed from the resin split off and the remaining protective groups removed.

709818/1082

26X9727

All amino acids used are included, if nothing otherwise stated, the naturally occurring L-isomers.

The invention thus provides carrier-bound peptides which are particularly useful for the production of peptides biological activity, especially carrier-bound peptides with (jy-CHp-O-Phe-Asn at one end of the amino acid chain, where (S) is the resin and Phe and Asn are the amino acids Phenylalanine and asparagine also mean processes for the production of these carrier-bound peptides.

The amino acid chains of the peptide resins according to the invention are identical to the amino acid chains of natural peptides with biological activity. It will also indicated other peptides with amino acid chains whose amino acids in terms of type and sequence of the amino acid chains of natural biologically active peptides are different, but peptides with biological Activity can be derived, as well as corresponding intermediates.

The total synthesis of the peptides according to the invention, natural or modified sequence, encompasses numerous Reactions in which numerous new peptide intermediates are also formed, which are described below with regard to their structural formula will be explained in more detail step by step using the following examples.

Manufacture of an insoluble resin

The insoluble resin abbreviated with the symbol (r) is a polymer material that is used in the peptide synthesis

709818/1082

The solvents used are insoluble but can be solvated and penetrated by them and act as an active receptor phase for the first amino acid, in the present case phenylalanine, can serve.

Practically preferred is the use of an insoluble polystyrene resin which is obtainable by the catalytic polymerization of styrene and divinylbenzene. That Resin selected as indicated is mixed with chloromethyl] -methy Ether and tin tetrachloride as a catalyst are chloromethylated according to the following reaction equation:

(T) + Cl-CH ₂ OCH ₃ (T) -CH ₂ -Cl + CH ₃ OH,

The reaction is detailed by the following Example explained.

example 1

1 kg of a polystyrene resin crosslinked to 2% with divinylbenzene and having a particle size of 0.055-0.075 mm (200-400 mesh) was washed three times with 2 liters of methylene chloride each time. Fine particles were removed by stripping off the methylene chloride from the bottom each time. The resin was then washed by suspending, stirring for 10 minutes and filtering through a glass frit using 2 l each of the following solvents: with 2 parts of tetrahydrofuran, with 2 parts of water, with 1 part of IN NaOH, with 2 parts of water, with 2 parts of dimethylformamide (DMF), with 2 parts of dioxane and with 3 parts of methanol. The washed resin was then dried ^{at 60 ° C. in vacuo.}

70 9818/1082

JH

500 g of the washed polystyrene resin were stirred with 5 1 chloromethyl methyl ether at room temperature] The temperature was then cooled in an ice-water bath to 0 - 5 ⁰ C lowered. Then 75 g of anhydrous tin chloride in 925 ml of ice-cold chloromethyl methyl ether were added and the mixture was stirred in an ice bath for 2 hours. The resin was filtered off through a glass frit and washed with 2 liters of each of the following solvents: 25% water in dioxane, 25 % 2-NHCl in dioxane, water / water and twice with methanol. The washed resin was ^{dried at 45-50 °} C. in vacuo. According to this procedure, the usual chloride content is 0.7 - 1.0 meq / g (meq = milliequivalent).

Phenylalanine esterification to the polystyrene resin

First, phenylalanine is bound to the polystyrene resin. The reaction takes place according to the following equation:

0-,

+ HOOC-C-CH ₂

NH

BA

0 H

f R J-CH ₂ -OCC-CH ₂ -NH

BA-HCl

Coupling reaction 1

709818/1082

Jß

in the formula mean:

(R) the polystyrene resin,
BA a suitable base such as triethylamine, diisopropylamine, diisopropylethylamine or an alkali metal salt

as

P is an amino protecting group such as amyloxycarbonyl (AMOC), o-nitrophenylsulfenyl (NPS) or preferably t-butyloxycarbonyl (BOC).

As can be seen from the above formula, this becomes t-butyloxycarbonyl-L-phenylalanine linked to the chloromethylated resin in the presence of an acid acceptor.

The reaction is shown in Example 2 below.

Example 2

50 g of the chloromethylated polystyrene resin prepared as described above with a chlorine content of 0.74 meq / g (yj meq chlorine) were stirred with 19.6 g of BOC-L-phenylalanine (74 meq) in 150 ml of absolute ethanol, whereupon 9, 77 ml of triethylamine (72 meq) were added; the mixture was refluxed with stirring for 24 hours. The mixture was then cooled, filtered through a Buchner funnel with a glass frit and poured on the frit with 500 ml parts of the following solvents

709818/1082

JN

washing: twice with denatured alcohol (alcohol denatured with 5 % methanol), twice with dioxane, twice with denatured alcohol, twice with water and twice with methanol. The resin was then dried at KO -45 ^° C. in a vacuum. The nitrogen analysis showed values between about 0.50-0.70 meq / g. After the BOC protective group had been split off with trifluoroacetic acid (described below), the free terminal amino group content of the resin was determined by titration to be about 0.38 meq / g.

Cleavage of the protective group

OHH

Θ «I I

-CH ₂ -OCCNP

CH "

1. Acid

2nd base

0 H

-CH ₂ -OCC-NH ₂

The resulting peptide-resin is referred to as Compound 1 .

The splitting off of the protective group from the amino function of the phenylalanine is done by using a suitable one Acid such as trifluoroacetic acid or hydrochloric acid. The resulting amine salt is then treated with neutralized with a strong organic base. The following example gives an embodiment of this method

709818/1082

game 5.

Example 3

6 g of the BOC phenylalanine resin according to Example 2 were introduced into the reaction vessel of a peptide synthesizer. The sample was twice with 40 ml of methylene chloride washed for 2 min. Then 40 ml of 50 ^ i Trifluoroacetic acid in methylene chloride was added and the Ge. mixed for 30 min to react. After filtration it was the resin three times with 40 ml of methylene chloride each time, twice with 40 ml of methanol each time and three times with 40 ml of chloroform each time each washed for 2 min. Then 40 ml of a 10 ^ solution of diisopropylamine in Chloroform neutralized for 5 min. The resin was then three times with 40 ml of chloroform and three times with 40 ml Washed methylene chloride.

coupling

0 H

R] -CHo-OCC-NH ₉ + AOOC-C-CHo-C-NH I II

0 0

OH H II HH -CH ₉ -OCCNCCNP + ι ι

CH-,

NH

I.
P.

or

709818/1082

a-θ-- JKo

26AS727

Θ | Ι η η υ

-CH ₀ -OCC-NH ₀ + HOOC-C-CH ₀ -C-NH 2 ι 2 II

P '

CHr

NH

O O

(H Il H H

Θ (η Ii η η

-CH ₉ -0-CCNCCNP ¹ II

In the formula:

P is an amino protecting group as described above,

A is an active ester such as p-nitrophenyl, o-nitrophenyl or pentachlorophenyl,

P 'is an amide protecting group such as benzhydryl, xanthydryl ο the like. and

CA is a coupling agent that forms peptide bonds such as diimides, azides, mixed anhydrides, and preferably dicyclohexylcarbodiimide (DCC).

The symbols (r), P, P ', A and CA mentioned have those previously defined in the following, including the claims Meaning.

Since formulas such as the above take up too much space, the reaction products are listed below in the corresponding abbreviated notation:

[R) -CHg-O-Phe-Asn-P

P ¹

in which Phe denotes phenylalanine and Asn asparagine residues and (R) ₅ P ¹ and P have the above meaning.

709818/1082

-HT-

This simplified nomenclature is used in the following for all reactions.

The splitting off of the protective group leads, as explained in connection with the phenylalanine resin, to one Product of the following structure:

(r) -CEL-O-Phe-Asn-NHo (Compound 2). V_y 2, 2

P '

As far as the inventors know, this is the first time this peptide resin has been produced, what represents an important step in the hormone synthesis of HPTH or the HPTH fragment.

The inventors also made the important finding that the completeness of the coupling reaction can be checked by the ninhydrin test (cf. E. Kaiser, R. Colescott, CD. Bossinger and P. Cook, Anal. Biochem. 54 (1970) 595-98). When the ninhydrin reaction is negative, the protective group can be split off from the resin and proceed to the following coupling reaction will. If the ninhydrin reaction fails, the coupling step is repeated until the Ninhydrin reaction is ultimately negative.

The following working examples relate to the coupling of asparagine.

example

About the deprotected phenylalanine resin 709818/1082

λ *

According to Example 3 with 3.5 meq amino groups, a solution of 7 mmol (about 100 % excess) BOC-L-ß-benzhydrylasparagine in 40 ml of methylene chloride was added. After 2 minutes, a solution of 7 ^m eq dicyclohexylcarbodiimide (DCC) was added and the mixture was stirred for 45 minutes. The product was filtered off and washed twice with 40 ml each of chloroform and methylene chloride. The ninhydrin reaction was performed on a 3-5 mg sample of the peptide resin reaction product and found to be negative. The protective groups according to Example 3 were then split off.

Example 5

2 g of phenylalanine resin were deprotected and neutralized as described above. Afterward 3 mmol of NPS-L-ß-benzhydrylasparagine, which were dissolved in 25 ml of methylene chloride, and 2 mmol Dicyclohexylcarbodiimide (DCC) added. The mixture was stirred for 1 h, filtered and washed with 2 parts of methylene chloride, Washed 2 parts of methanol and 3 parts of methylene chloride.

Example 6

Instead of the NPS derivative in Example 5, the AMOC derivative can be used using the same equivalent amounts can be used with the same results.

709818/1082

Peptide synthesis

In the following table 1 are the corresponding consecutive Reaction steps 2-34 under the designation of the respective link position in the chain Mention of the amino acid reactants synthesized in each step and the preferred protecting groups.

Table 1

Reaction position to synthesized No. No. Amino acid Amino acid group with preferred protecting group

2 33 . Asparagine 3 32 Histidine 4th 31 Valine 5 30th Aspartic acid 6th 29 Glutamine 7th 28 Leucine 8th 27 Lysine

10

11

26th

25th
24

Lysine

Arginine

Leucine BOC-L-ß-Benzhydrylasparagine

BOC-L-im-carbobenzyloxy-L-histidine

BOC-L-VaIin

BOC-L-ß-Benzylaspartate

BOC-L-glutamine-p-nitrophenyl ester

BOC-L-leucine

BOC-E-2-chlorocarbobenzyloxy-L-lysine in 10 $ DMF for solubility

BOC- ε-2-chlorocarbobenzyloxy-L-lysine in 10 % DMF for solubility

BOC-L-Tosylarginine in 20 % DMF for solubility

BOC-L-leucine

709818/1082

13th

34- -

ίο

(Tabel I ₁ Continuation)

12th 23 Tryptophan 13th 22nd Glutamic acid 14th 21 Valine 15th 20th Arginine 16 19th Glutamic acid 17th 18th Methionine 18th 17th Serine 19th 16 Asparagine 20th 15th Leucine 21 14th Histidine

Lysine

BOC-L-Tryptophan in 10 % DMF for solubility

BOC-L - ^ - benzyl glutaminate BOC-L-valine

BOC-L-Tosylarginine in 20 % DMF for solubility

BOC-L- 7-benzyl glutamate BOC-L-methionine BOC-0-Benzyl-L-serine BOC-L-ß-Benzhydrylasparagine BOC-L-leucine

BOC-im-carbobenzyloxy-L-histidine

BOC- £ - chlorocarbobenzyloxy-L-lysine in 10 ^ DMF for solubility

23 12th Glycine BOC glycine 24 11 Leucine BOC-L-leucine 25th 10 Asparagine BOC-L-ß-Benzhydrylasparagine 26th 9 Histidine BOC-im-Carbobenzyloxy-L-
histidine 27 8th Methionine BOC-L-methionine 28 7th Leucine BOC-L-leucine 29 6th Glutamine BOC-L-glutamine-p-nitrophenyl
ester 30th 5 Isoleucine BOC-L isoleucine 31 4th Glutamic acid BOC-L-7-benzyl glutamate 32 3 Serine BOC-0-benzyl-L-serine 33 2 Valine BOC-L-valine 34 1 Serine BOC-0-benzyl-L-serine

709818/1082

Each subsequent reaction step of linking a further amino acid group proceeds in the same way Manner as described in connection with the linkage of asparagine in reaction 2 (see example 4), by coupling the previously obtained peptide resin with the following amino acid in a protected state, the protective group is split off from the newly coupled peptide and neutralized. In detail will be The following reaction steps were carried out in each case:

Coupling:

7 mmol of the unique BOC amino acid (0.43 equivalent excess in 40 ml of methylene chloride or DMF mixture, if necessary) _;

7 mmol of dicyclohexylcarbodiimide (coupling agent) in 15 ml of methylene chloride, reaction time 45 min _; twice washing with 40 ml of chloroform each time, each time for 2 min and

washing twice with 40 ml of methylene chloride each time, 2 min each time;

Cleavage of the protective group:

twice washing with 40 ml methylene chloride each time, 2 min each time _?

50 % trifluoroacetic acid in methylene chloride, 5 min, 40 ml (after reaction no. 12, 1 <fo 2-mercaptoethanol or ethanedithiol is added to the 50% trifluoroacetic acid in methylene chloride) _?

709818/1082

Wl

(Continuation:)

washing three times with 40 ml of methylene chloride each time, 2 min each time,

twice washing with 40 ml of methanol each time, each time 2 min _} and

washing three times with 40 ml of chloroform each time, 2 min each time;

Neutralization:

twice each 40 ml of 10 % diisopropylamine in chloroform, each 5 min and

washing four times with 40 ml of chloroform each, 2 min each time.

The steps of coupling, splitting off the protective groups and neutralization in reactions 3 - 3 ^ correspond as described in reaction 2 with the exception of the following deviations:

As already stated, compound 2 originating from reaction no. 2 shows after cleavage of the protective group and neutralization on the following structure:

(r) -CH ₀ -O-PtIe-ASn -NH ₀ ;

W d , d.

P ¹
after reaction 3 there is compound 3:

-CH ₀ O-PhC-ASn-HiS-NH ₀ ; 2,, «d

P 'W

709818/1082

after. Reaction 4 is compound 4:

-CH ₀ -O-Phe-Asn-HiS-VaI-NH ₀ ; d Il ²

P 'W
after reaction 5 there is compound 5:

R) -CH ₀ -O-Phe-Asn-His-Val-Asp-NHo II IP ¹ W Bz

In reaction _3a, where histidine is coupled in position 32, carbobenzyloxy (CBZ) is preferably used to protect the itnidazole group, but tosyl or dinitrophenyl (DNP) can also be used as a protective group for this in the same way. The abbreviation W denotes CBZ, Tosyl or DNP.

In reaction 5 * where aspartic acid is coupled in position 30 benzyl or a benzyl derivative is preferably used as the protective group. Under the abbreviation Bz is accordingly understood to be benzyl or a benzyl derivative.

Are under benzyl derivatives in the sense deluded here Derivatives of the benzyl group such as halogenated benzyl, alkylated benzyl or alkoxylated benzyl or the like. Understood. Such derivatives are common in peptide chemistry, which is why a more detailed explanation is not necessary.

The synthesis is carried out in the manner indicated up to the linkage of GIn in position 29. In this

7 09818/1082

Position, DCC can only be used as a coupling agent if the glutamine has a suitable protecting group such as benzhydryl or xanthydryl; the groups mentioned can either be bound to it or protective catalysts can be added to the corresponding solutions. Caused without such protection DCC is a side reaction that consumes some of the glutamine. Alternatively, glutamine can be unprotected Form can be coupled as an active ester.

The carrier-bound peptide freed from protective groups '' hereinafter referred to as peptide resin for short) is marked with a active esters of glutamine such as p-nitrophenyl ester, the o-nitrophenyl ester or the pentachlorophenyl ester moved or stirred.

The corresponding coupling step is described below Example 7 explained in more detail.

Example 7

That of the compound 5 according to reaction 5 corresponding

Peptide resin (after splitting off the protective group and neutralizing
sation) was three times with / 40 ml of dimethylformamide each

Washed for 2 min. 12 mmol dissolved in 40 ml of dimethylformamide BOC-L-glutamine-p-nitrophenyl ester were 20 h with the resin shaken and then with 3 parts of dimethylformamide,

Washed 3 parts of methanol and 3 parts of methylene chloride. The glutamine in position 6, reaction 29, is coupled in the same way.

709818/1082

The coupling in position 16 is followed by the usual splitting off of the protective groups and neutralization at; this results in a peptide resin (compound I9) of the following structure:

i -O-Phe-Asn-His-Val-Asp-Glti-Leu-Lys-Lys-Arg-Leu-Trp-Glu-Val-Arg-Glu-

² II Il III '']

j. i Bz P ¹ VVT B ₂ TB ₂

-Met-Ser-Asn-NH2

B ₂ P ¹

(Compound 19).

When linking lysine in reaction no. 8, position 27, 2-chlorocarbobenzyloxy (Cl-CBZ) is preferably used as the B-amino protective group, but 2-bromocarbobenzyloxy or 2- dichlorocarbobenzyloxy can also be used in the same way.

The designation V is used to denote verx ^ ends, that the E-amino protecting group consists of one of the above groups.

For coupling the amino acid arginine in reaction no. Or position 25 is used as a protective group to protect the Guanidino puncture preferably the tosyl group (p-toluenesulfonyl group) used, but a nitro group can also be used in the same way; as a short name the symbol T is used for this, which means tosyl or nitro.

The symbols T and V have like the other abbreviations in all places used including the

709818/1082

Claims the same meaning.

The ninhydrin test is carried out after each coupling reaction and before the protective groups are split off from the peptide resin. If the test is positive, the coupling reaction carried out last is repeated. When the test is negative, vri.rd for splitting off protective groups from the peptide resin passed over.

After the connection of the serine in reaction no. J4 in position 1 according to the procedure and sequence described above and after splitting off the protective groups and neutralizing the coupled peptide resin, compound ~ $ K with the following structure is obtained:

Ö-CHo-O-Phe-Asn-His-Val-Asp-Gln-Leu-Lys-Lys-Arg-Leu-Trp-Glu-Val-Arg-Glu-Met- Il Il! III ''

P »W Bz P ¹ VVT Bz T Bz

-Ser-Asn-Leu-His-Lys-Gly-Leu-Asn-His-Met-Leu-Gln-Ile-Glu-Ser-Val-Ser-N ^

Il Il Il '! I I

_Bz pt wv P ¹ WP ¹ Bz Bz Bz

(Link

This peptide resin is then treated further to split off the resin and the remaining protective groups. The resin as well as any remaining protecting groups can suitably by treatment with anhydrous Ground hydrogen fluoride removed. The reaction equation for this is:

709818/1082

(R Vc ^ -O-Phe-Asn-HIs-Val-Asp-Gln-Leu-Lys-Lys-Arg-Leu-Trp-Glu-Val-Arg-Glu-

^ - ^ Il Ii ill ι Ii

P ¹ W BzP ¹ VVT Bz T Bz

-Met-Ser-Asn-Leu-His-Lys-Gly-Leu-Asn-His-Met-Leu-Gln-Ile-Glu-Ser-Val-Ser-NH

Il Il Il t Il ι

Bz P ¹ WV P ¹ WP ¹ Bz Bz Bz

(Response 35) HF

Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe 21 22 23 24 25 26 27 28 29 30 31 32 33 34 (connection 35)

Example 8 below illustrates this cleavage reaction.

Example 8

2 g of compound 32 along with 2 ml Anisole was placed in a vessel made of KeI-F, whereupon 10 ml of anhydrous hydrogen fluoride were added by distillation became. The mixture was then stirred at 0 ° C. for 1 h. The hydrogen fluoride was then removed by vacuum distillation removed and the residue washed four times with ethyl acetate and then with glacial acetic acid extracted. The acetic acid extract was then lyophilized to a fluffy white powder. Through this procedure the peptide is split off from the resin, all protective groups on the amino acids being removed at the same time will.

70981 8/1082

Example 9

In the same manner as in the previous examples, a 1-34 peptide resin was prepared in which the L-serine in position 1 by treating compound 33 with BOC-O-Benzyl-D-serine instead of BOC-0-Benzyl-L-serine was replaced by D-serine. After the cleavage of the resin and the remaining protective groups lay the following reaction product:

D-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

(Compound 36).

This analog possessed by the chick hypercalcemia test (See J.A. Parsons, B. Reit and CJ. Robinson, Endocrinology 92 (1973) W) biological activity.

cleaning

After gel filtration on Biogel P-6 (manufacturer Biorad), the crude human 1-34 parathyroid hormone peptide (HPTH (1-34)) was chromatographed on carboxymethyl cellulose (CMC) (Whatman CM52) using a linear gradient of ammonium acetate buffer. After desalting on polyacrylamide gel, the homogeneity of the synthetic peptide was checked by thin layer chromatography on cellulose (Brinkmann Celplate-22, Eastman 6θβ5) and silica gel plates ( Merck). The amount of sample applied was 30 / Ug in 5 μl of 0.1 IM acetic acid. The following solutions were

709818/1082

solvent systems used: R ^ n-butanol / acetic acid / water 4: 1: 5; R “ethyl acetate / pyridine / acetic acid / water 5: 5: 1: 3; R ° n-butanol / pyridine / acetic acid / water 15: 10: 3: 12; R _f n-butanol / acetic acid / water / ethyl acetate 1: 1: 1: 1. The peptide spots were visualized by spraying the plates with Ehrlich's reagent and 0.5 % ninhydrin in ethanol. The purified synthetic HPTH (1-34) peptide gave a single spot with R ~ (cellulose, Brinkman) 0.19; R _f (silica) 0.11; R ° (silica) 0.17; R ° (cellulose, Brinkman) 0.40; R ° (cellulose, Eastman 6065) 0.66 and R "(cellulose, Brinkman) 0.48.

The biological activity of the synthetic HPTH- (1-34) in vitro based on the rat kidney adenylate cyclase test and the chick hypercalcemia test are shown in Table 2 below. For comparison purposes are also corresponding data on native bovine PTH - Cl-84) [βΡΤΗ- (1-84ϊ (native)] and synthetically produced bovine PTH- (1-34) [bPTH- (1-34) J as well as native human PTH- (1-84) reported.

Table 2

Biological activity of synthetic and natural parathyroid hormones

in vitro in vivo

Rat kidney adenylate chick hypercalcemia

cyclase [mRC u / mg] [MRC u / mg]

HPTH- (1-84) (native) 350 unknown

HPTH - (1-34) (cleaned,

Compound 35) ^ ^{υυ ρΗυυ}

BPTH- (1-84) (native) 3000 2500

BPTH- (1-34) 5 ¹ ^ OO 7700

709818/1082

3o

From the examples given it can be seen that the synthesis of PTH peptides with biological activity with the method according to the invention as well as the corresponding peptide intermediate products in remarkable can be carried out in a favorable manner, which in view of the state of the art in technical peptide synthesis was completely unexpected.

The method according to the invention is not limited to the synthesis of PTH peptides, but can be e.g. also transferred to other reactions on solid supports in which similar sequences are built up.

709818/1082

Claims (12)

Expectations 1. Carrier-bound peptide of structure NH _o -Asn-Phe-0-CH _o - (R, P ¹ ) = insoluble polystyrene resin and P '= xanthydryl or benzhydryl. 2. Carrier-bound peptide of structure NH ₂ -VaI-HiS-ASn-PhC-O-CH ₂ - (r) W P ' ) = insoluble polystyrene resin, P '= xanthydryl or benzhydryl and W = carbobenzyloxy, tosyl or dinitrophenyl. 3. Carrier-bound peptide of structure NHo-Asp-Val-His-Asn-Phe-O-CEL 2 ,,, Bz W P 'with: 709818/1082 (RJ = insoluble polystyrene ^{resin, P 1} = xanthydryl or benzhydryl, W = carbobenzyloxy, tosyl or dinltrophenyl and Bz = benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitro benzyl or benzhydryl. 4. Carrier-bound peptide of structure N ^ -Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asft-Leu-Gly-Lys-His-Leu-Asn I Il I I I I I I Bz Bz Bz P ¹ WP 'VW P ¹ Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn Il I III I 'I' Bz Bz T Bz TVV P 'Bz WP ¹ = insoluble polystyrene resin, P '= xanthydryl or benzhydryl, T = tosyl or nitro, V = 2-chloro-carbobenzyloxy, 2-bromocarbobenzyloxy or 2,4-dichlorocarbobenzyloxy, Bz = benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl or benzhydryl and W = carbobenzyloxy, tosyl or dinitrophenyl. 5. peptide of structure D-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn- - Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe, 709818/1082 6. Process for the synthesis of a carrier-bound peptide, characterized in that P-Bz-L-serine is coupled on NHo-Väl-Ser-Glu-lie-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser- | i Ii IiII Bz P ¹ WP ¹ VW P ¹ Bz -Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-O- Il t III I I "I I Bz T Bz TVV P ¹ Bz WP ¹ -Θ ) - insoluble polystyrene resin, Bz = benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl or benzhydryl, V / = carbobenzyloxy, tosyl or dinitrophenyl, P ¹ = xanthydryl or benzhydryl, V = 2-chlorocarbobenzyloxy, 2-bromocarbobenzyloxy or 2,4-dichlorocarbobenzyloxy, T = tosyl or nitro and P = t-butyloxycarbonyl, amyloxycarbonyl or o-Nitrophenylsulfenyl as amino protective groups. 7. A method for the synthesis of a carrier-bound peptide, characterized in that P-Bz-D-serine is coupled on 709818/1082 -28-- NHo-Val-Ser-Glu-Ile-Gln-Leu-Met-Hie-AHn-Leu-Cly-Lys-Hie-Leu-Asn-Ser- Il I Il I I I I Bz Bz P ¹ WP ¹ VW P ¹ Bz ■ Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-O- Il I I I I I I Il Bz T Bz TVV P ¹ Bz WP ¹ (r) = insoluble polystyrene resin, Bz = benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl or benzhydryl, W = carbobenzyloxy, tosyl or dinitrophenyl, P ¹ = xanthydryl or benzhydryl, V = 2-chlorocarbobenzyloxy, 2-bromocarbobenzyloxy or 2,4-dichlorocarbobenzyloxy, T = tosyl or nitro and P = t-butyloxycarbonyl, amyloxycarbonyl or o-Nitrophenylsulfenyl as amino protective groups. 8. The method according to claim 6 or γ _}, characterized in that the reaction product is further treated with anhydrous hydrogen fluoride to split off the groups (r) -CHg, Bz, P ¹ , T, V and W. 9. Process for the synthesis of a carrier-bound peptide, characterized in that InIH ₂ -Phe-O-CH ₂ - ( ¹ R] 709818/1082 P-Asn P ¹ is coupled to a carrier-bound peptide of the structure P-Asn-Phe-O-CHg-P ¹ (r) = insoluble polystyrene resin, P '= xanthydryl or benzhydryl and P = t-butyloxycarbonyl, amyloxycarbonyl or o-Nitrophenylsulfenyl as amino protective groups. 10. The method according to claim 9, characterized in that the protective group P is also split off from the peptide will. 11. The method according to claim 10, characterized in that the reaction product of claim 10 with P-L-His to a carrier-bound peptide of structure W P-His-Asn-Phe-O-CHo- (R. I I <- W P ' is coupled with W = carbobenzyloxy, tosyl or dinitrophenyl. 709818/1082 12. The method according to claim H _3, characterized in that the protective group P is also split off from the peptide. 709818/1082