DE1212980B - Process for the preparation of peptides containing lower alpha, omega-diamino-alkanoic acids - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

InI. α .:

C07c

German class: 12 q- 6/01

Number: 1212980

File number: C 27909IV b / 12 q

Filing date: September 11, 1962

Opening day: March 24, 1966

The present invention is a new process for the production of peptides which a lower α, ω-diamino-alkanoic acid, in particular with 4 to 6 carbon atoms, such as lysine, ornithine or γ-diaminobutyric acid. For the sake of synthesis natural peptides use the L-form of the amino acids.

In the synthesis of peptides, which α, ω-diaminoalkanoic acids contain, it is necessary to protect the two amino groups temporarily or from to selectively release only the α-amino group from the protected groups. The selective splitting off of the A-amino protective group has not been able to be carried out in many cases, as with the known Combinations of protective groups in «- and ω-positions at the same time also prevent the undesired splitting off of the ω-amino protecting group occurred.

It has now been found that in the synthesis of ", cu-diamino-alkanoic acids containing peptides co-amino group in an advantageous manner through the phthalyl radical and the a-amino group through the tert-Butyloxycarbonylgruppe is protected and that the tert-butyloxycarbonyl group is obtained from the peptides obtained by condensation in a known manner removed in a known manner by acidic agents and at the end of the synthesis the phthalyl group in Cleaves the presence of hydrazine in a weakly acidic medium.

The condensation of the α, ω-diamino-alkanoic acid or a peptide containing it with a further amino acid or a peptide is carried out in a manner known per se.

For example, one of the amino acid or peptide molecules in the form of an ester can be linked to a further amino acid or peptide molecule containing a protected amino group in the presence of a condensing agent such as a carbodiimide or a phosphorous acid ester halide, or the amino acid ester can be or peptide esters with free amino group an amino acid or a peptide with activated carboxyl group (and protected amino group), z. B. an acid halide, azide, anhydride, imidazolide, isoxazolide (e.g. from N-ethyl-5-phenylisoxazolium-3'-sulfonate [see Woodward et al., J. Am. Chem. Soc, 89,196I ₅ p. 1011]), or an activated ester such as cyanomethyl ester or carboxymethyl thiol ester. Conversely, an amino acid or a peptide with a free carboxyl group (and a protected amino group) can also be combined with an amino acid or a peptide with an activated amino group (and a protected carboxyl group), ζ. B. a phosphite amide to be reacted.

Process for the production of lower
α, ω-diamino-alkanoic acids containing peptides

Applicant:

CIBA Aktiengesellschaft, Basel (Switzerland)

Representative:

Dr.-Ing. Dr. jur. F. Redies,

Dr. rer. nat. B. Redies

and Dr. rer. nat. D. Türk, patent attorneys,

Opladen, Rennbaumstr. 27

Named as inventor:

Dr. Robert Schwyzer, Riehen;

Peter Sieber, Basel (Switzerland)

Claimed priority:

Switzerland of September 13, 1961 (10599),
of March 14, 1962 (3,069)

If the α, ω-diaminoalkanoic acid or a peptide containing it is condensed onto the amino end of an amino acid or a peptide, the N «-BOC-N ^m -phthalyl derivative is used, e.g. B. N "-BOC-N <° -phtharyl-lysine. If, on the other hand, the diaminoalkanoic acid or a peptide containing it is condensed onto the carboxyl end of an amino acid or a peptide, the first must have a free α-amino group; the BOC group must first be split off from an N ^ BOC-N ^ -phthalyl derivative.
The derivatives of α, ω-diaminoalkanoic acids used as starting materials, in which the amino groups are protected in the manner mentioned, are new. Starting from known N ^ra phthalyl derivatives, z. B. the N ^s -phthalyl-lysine, they cannot be produced. The starting materials, ζ. B. the new N "-tert.-Butyloxycarbonyl-N ^e -phthalyl-L-lysine (BOC-Lys (Pht) -OH), can be synthesized as follows:

The carbobenzoxy group (Z) of the well-known BOC-Lys (Z) -OH is produced by catalytic hydrogenation cleaved. The new BOC-lysine obtained is in an alkaline medium with N-carbethoxy-phthalimide implemented, whereby the BOC-Lys (Pht) -OH is obtained in practically quantitative yield. In analog

609 539 / 41S

ι EU

Pm O

υ ο

PQ

PU

Ό O

PQ

Yy.

J? 3

r U O

pq

j
Ό
O
PQ DCCI J HO " I: BOC- ο ο
ι ι £ I.
U
O
PQ

Ph

pq.

3 .5 3

pq

'Ph

do

Ph O I. U ■ + -> ys VaI Ty I— *
O U Pm 8th ? - <

Ph

O O

pq

Ph-

Ph

O O

pq

O O pq

Ü O

pq

5 = 1 <

το

• c

i § +

em

PQ

PQ

S-

ζ-

ζ-

P * ~

ζ-

Z-

υ ο

O
ι Oil
§- O
I. Per d Per Tyr Pht Tyr VaI VaI ÖD Lys VaI VaI Per Per Arg Arg Lys

ι X

Ph O

I. Pht I. Per VaI . d VaI 'OM Lys 13th ai- 13th Per Per Arg -I -I -I

U O

Ph

S-

Z-

S-

Ph-

-I -I

-3

OI
O
z- -I Pht O Ti K X I. I. O O Ü Lys I. ι O I. d -ο
<Ph- Lys O . I. PQ O O PQ O PQ

The other N ^ -BOC-N * "- phthalyl-diaminoalkanoic acids can also be used produce.

As is known, a phthalyl radical can easily be split off by alkaline treatment, in particular with hydrazine. Such a cleavage can, however, with many peptides, so z. B. with corticotropin and κ- or / 3-MSH, which are particularly sensitive to alkali, do not perform. On the other hand, the phthalyl radical is very resistant to strong acids. It was

ίο now surprisingly found that the Phthalyl radical advantageously in a weakly acidic medium at a pH between 4 and 7 in the presence can be split off from hydrazine. The solutions are made by combining hydrazine with an organic or inorganic acid or by dissolving a hydrazine salt in aqueous or aqueous-organic solution. The acids can be, for. B. Hydrochloric acid, ■ sulfuric acid, phosphoric acid, etc., or acetic acid, propionic acid, phthalic acid, benzoic acid etc., can be used.

In contrast, the tert-butyloxycarbonyl radical can be used only split off in strongly acidic media at a pH value below 4. It can be strong inorganic or organic acids can be used.

The different behavior of the phthalyl and tert-butyloxycarbonyl radicals towards alkaline, weakly and strongly acidic agents is of great advantage in peptide synthesis. While the N ^ phthalyl ^{radical of the N a} -BOC-N ^ffl -phthalyl peptide must remain intact in the synthesis of a long-chain peptide through several process steps, even up to the final stage of the synthesis, the tert-butyloxycarbonyl radical can be passed at any suitable synthesis stage Separate treatment with strong acids. If desired, the tert-butyloxycarbonyl radical can also be left in place and the phthalyl radical can be cleaved off in a weakly acidic medium of a hydrazine salt.
So z. B. produce the partial sequence of amino acids 15 to 24 of corticotropin given below by using the new lysine derivative, namely Na-BOC-Ne-phthalyl-lysine, in excellent yield.
An advantageous implementation of the synthesis of the part-sequence mentioned is recorded in scheme X. In this synthesis it is shown at the same time that p-phenylazobenzyl alcohol is very suitable for esterifying or protecting a carboxyl group. The amino acid or peptide esters obtained are colored and thus facilitate the isolation and purification of these intermediates, e.g. B. in partition chromatography or multiplicative distribution. The colored carboxyl protective group can then be removed by hydrazinolysis, hydrolysis or catalytic hydrogenation.

BOC means a tert-butyloxycarbonyl group, Py pyridine, TF trifluoroacetic acid, DCCI dicyclohexylcarbodiimide, PAB p-phenylazobenzyl and »acc. App. " the peptide linkage method of "mixed anhydrides".

The linkage of a lysine derivative with a peptide by the activated ester method is illustrated in Example 3 [condensation of BOC-Lys (Pht) pentachlorophenyl ester with Arg (NO ₂ ) -Arg

6s (NO ^ -Pro-Val-Lys-CPüQ-Val-Tyr-Pro-OPAB].

The new process also offers advantages in the synthesis of the α-melanophore-stimulating hormone (a-MSH) of the formula «-Acetyl-L-seryl-L-tyrosyl-

Scheme 2

QC ₄ H ₉ (tert.)

BOC-

Ser Tyr Ser Met GIu His Phe Arg Try GIy

Pht

-OH BOC-

Lys

-OH BOC-

Pht

BOC- Lys

Pht

H-

Lys

Per -OH Z- VaI

BOC-

Per

Per

Per

Per

Per

-OH

NH ₃ I according to anhydride Z-

VaI

-NH ₉

H ₂ ψ (Pd) in acetic acid H-

VaI

-NH ₂ , OBLCOOH

ψ according to anhydride

VaI

-NH _a

j TF

VaI -NH ₂ , CF ₃ COOH

I Amberlite IRA-400 ν (acetate form)

VaI

-NH ₂ , CH ₃ COOH

ψ according to anhydride

VaI -NH ₂

φ HCl in ethyl acetate

VaI

-NH ₂ , HCl

Ph

CO

OO

00

Γ U O

ffi

O O O

X OOU

si

CS

ta

Ph -

_t ■ *

S?

Ph τη

OO

Tyr

00

ffl

OO

O O

L-seryl-L-methionyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycyl-L-lysyl-L-prolyl-L-valine amide. This tridecapeptide was developed by St. Guttmann and R.A. Boissonnas synthesized (HeIv. Chim. Acta, 42, p. 1257 [1959]), but the cleavage of the protective groups presented great difficulties. The synthesis described here, in which the amino group of the lysine is protected by the phthalyl radical is, however, runs very smoothly. Scheme 2 ίο shows the course of the synthesis in which the known Decapetide derivative BOC - Ser - Tyr - Ser - Met - GIu (O-tert-butyl) -His-Phe-Arg-Try-Gly-OH (see patent application C 24191 IVb / 12q [Deutsche Auslegesshrift 1205 546]) with the tripeptide derivative N * -Phtha-

W 15 lyl-L-lysyl-L-prolyl-L-valine amide is linked.

^ The invention is illustrated in the examples below

described in more detail. The temperatures are given in degrees Celsius.

example 1

20th

a) BOC-Lys (Pht) -OH

41g BOC-Lys (Z) -OH (Literature: G.H. Anderson and A.C. McGregor, Journ. At the. Chem. Soc, 79, p. 6180 [1957]) are in 400 ml of 95% Hydrogenated methanol in the presence of 4.1 g of 10% palladium carbon. After filtering off the catalyst the filtrate is evaporated in vacuo. The remaining foam is in 100 ml of absolute Dissolved ethanol; when the solution is stored, the BOC-L-lysine crystallizes out. Total will be 24 g = 90% obtained. F. 204 to 205 ° (with decomposition). The substance can be made from water — acetone be recrystallized. It is uniform in terms of paper chromatography. The melting point changes not. 17.6 g of the above BOC-L-lysine are in 70 ml of water dissolved with 7.6 g of anhydrous sodium carbonate. 19.7 g of N-carbethoxyphthalimide are added with stirring added and stirred for a further 30 minutes. The solution is filtered clear, cooled to 0 °, with 2N hydrochloric acid acidified to pH 2 and extracted with ethyl acetate. The ethyl acetate extracts are made with 120 ml Saturated sodium hydrogen carbonate solution extracted, this in turn acidified with 2N hydrochloric acid and extracted with ethyl acetate. From the ethyl acetate extracts are obtained after washing, drying and Evaporation 26.6 g = 99% of theory of a colorless vitreous product. The connection is ethnographically uniform.

b) Pro-OPAB, HCl

20.9 g BOC-L-proline and 22.7 g p-phenylazobenzyl alcohol are dissolved in 200 ml of pyridine, treated with 22 g of dicyclohexylcarbodiimide at 0 ° and then over Left to stand overnight at room temperature. After adding a few milliliters of glacial acetic acid, the mixture is cooled to 0 ° and the dicyclohexylurea sucked off. After the pyridine has evaporated, the filtrate is dissolved in ethyl acetate dissolved and made up to neutral product with 0.5N hydrochloric acid and sodium hydrogen carbonate solution.

is working. About 40 g of a red oil are obtained.

This is dissolved in 100 ml of absolute ethyl acetate and mixed with 500 ml of 3N hydrochloric acid in ethyl acetate. After V ₂ hours, the mixture is evaporated to dryness in vacuo at 40 °. The residue is dissolved in 500 ml of chloroform and filtered through a column of 1 kg of silica gel. An impurity can be eluted with chloroform, then the substance sought is washed out with chloroform + 10% methanol. Obtain

609 539/415

11 12

34 g of Pro-OPAB, HCl. These are made up of abso-, LvsiPhtf-Vni-Tvr-Pro-OPAB

lutemethanol recrystallized. Yield: 26.0g == 777 ₀ ^g) ^ ⁸ ^^ ^{Val 1} ^ ^{Fro ÜJ? AJJ}

the theory; F. (177) 180 °. 9.08 g of BOC tetrapeptide are used as under e)

Treated and worked up trifluoroacetic acid. Ausc) Tyr-Pro-OPAB, HCl _{5 yield: 8j} i _g Lys (Pht) -Val-Tyr-Pro-OPBA = 100%

.. 1.39 g of Pro-OPAB, HCl are in 10 ml of water according to theory.

Thin layer chromatogram:

washed neutral and evaporated in vacuo at 40 °. Chloroform-Acetone 7: 3 ... Uniform Rf 0.16

The residue and 1.13 g of BOC-Tyr-OH are dissolved in 1 ° benzene-acetone 1: 1 uniformly Rf 0.42

Dissolved 20ml acetonitrile and 1 ml dimethylformamide,. _Ώ _ _,. ,. _{1 ηη} . _Ό

and 0.91 g of dicyclohexylcarbodiimide were added at 0 °. ^h > BOC-VaI-LyS (PM) -VaI-TVr-PrO-OPAB

After standing overnight at 0 °, the dicyclohexyl from 3.5 g of BOC-VaI-OH and 8.1 g of Lys (Pht) -Val-

urea suction filtered, the filtrate concentrated, in vinegar; Tyr-Pro-OPAB are described under d)

ester added and with O, 5N hydrochloric acid and> 5 benen method 8.8 g of BOC pentapeptide = 88% of the

Sodium hydrogen carbonate solution shaken out. Get the theory.

The neutral product obtained is treated with hydrochloric acid in vinegar _ ..,. ,,,

ester freed from the BOC group The hydrochloride thin-layer chromatogram:

crystallizes from methanol-ether. Yield: chloroform-acetone 7: 3 ... Uniform Rf 0.36

1.68 g = 82% of theory; M.p. 204 ° (with decomposition). 20 Benzene-Acetone 1: 1 Uniform Rf 0.65

BOC-Val-Tyr-Pro-OPAB i) Val-Lys (Pht) -Val-Tyr-Pro-OPAB

9 g of BOC-VaI-OH, dissolved in 90 ml of absolute tetra- The above 8.8 g of BCC pentapeptide ester is used with

hydrofuran and 5.7; ml of triethylamine, are described with -10 trifluoroacetic acid according to the under ^ e)

to -15 ° with 5.0 ml of isobutyl chlorocarbonate, as benen method 8.2 g Val-Lys (Pht) -Val-Tyr-Pro-

After 15 to 20 minutes, 17.25 g of OPAB = 10% of theory are obtained at the same time. Tyr-Pro-OPAB, HCl, dissolved in 120 ml of absolute

Dimethylformamide and 4.7 ml of triethylamine, in 45 ml of thin-layer chromatogram:

added dropwise to absolute tetrahydrofuran. Then chloroform-acetone 7: 3 ... Uniform Rf 0.14

the mixture is stirred for a further 1 hour at 0 °, then overnight in 3 ° benzene-acetone 1: 1 Uniform Rf 0.33

Left the refrigerator. The mixture is

concentrated in ethyl acetate and taken up at 0 ° with _{k) B} OC-Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB
0.5N hydrochloric acid and sodium hydrogen carbonate solution

shaken out. The neutral product obtained is 3.3 g of BOC-Pro-OH and 8.2 g of Val-Lys (Pht) -Val-

recrystallized from methanol-water. Yield: 35 Tyr-Pro-OPAB are described analogously to under d)

20.15 g BOC-Val-Tyr-Pro-OPBA = 88% of the Theobe method implemented and worked up. Yield:

rie; 106-108 °. . 9.05 g BOC-Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB-

Thin-layer chromatogram: uniform in chloro- = 91% of theory.

form — acetone 7: 3; Rf 0.54. _ ..,. ,,,

₄₀ thin layer chromatogram:

e) Val-Tyr-Pro-OPAB Chloroform-Acetone 7: 3 ... Uniform Rf 0.25

17.0 g of BOC-Val-Tyr-Pro-OPBA become Rf 0.49 under benzene-acetone 1: 1

Cooling with cold water with 50 ml trffluoroacetic acid-chloroform-methanol 9: 1 uniformly Rf 0.75 acidic poured over and shaken until dissolved. The solution is kept for 5 minutes at room temperature, 45 _{% p ro} -Val-Lys (Pht) -Val-Tyr-Pro-OPAB
then evaporated in vacuo at room temperature.

The oil is dissolved in chloroform, once with water 9.05 g BOC-Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB

extracted, then the chloroform solution at 0 ° as described under e) with trifluoroacetic acid

saturated sodium hydrogen carbonate solution stirred until treated. Yield: 8.65 g Pro-Val-Lys (Pht) -Val-

there is no longer any evolution of carbon dioxide. 50 Tyr-Pro-OPAB = 100% of theory.

The chloroform layer is separated with sodium. ,. ,

sulfate-dried and evaporated to dryness. Yield: thin-layer chromatogram:

14.8 g Val-Tyr-Pro-OPBA = 100% of theory. Chloroform-Methanol 9: 1 Uniform Rf 0.38

Thin-layer chromatogram: ₅₅ m) Pro-Val-Lys-Val-Tyr-Pro-OH

Chloroform-Acetone 7: 3 ... Uniform Rf 0.2 Pro-Val-Lys- (Pht) Val-Tyr-Pro-OPAB is by

Benzene-Acetone 1: 1 Uniform Rf 0.45 Hydrogenation in the presence of palladium-carbon, in

^ τ, ηη τ rvu + s _lr , τ r> γ «>λγ> Pro-Val-Lys (Pht) -Val-Tyr-Pro-OH transferred,

f) BOC-Lys (Pht) _{-Val-Tyr-Pro-Pro} OPAB _{500 mg of this} ^V d _{will uktes} n for the purpose of cleavage

From 7.4 g of BOC-Lys (Pht) -OH is, as under d), 60 of the phthalic group with 5 ml of a methanolic erasure, the mixed anhydride prepared and solution that is 2 molar of hydrazine monoacetate (pH 6.5)

this implemented with 6.43 g of Val-Tyr-Pro-OPAB. stored at 50 ° for 15 hours. The free

Yield: 9.08 g BOC-Lys (Pht) -Val-Tyr-Pro-OPAB Hexapeptide is paper chromatographically and electronically

= 87% of theory. trophoretically checked and consistently found. From the

• η »·· ι. · Τ. * T. * ⁶ S-free peptide is a quantitative

Thin-layer chromatogram: _tative amino acid analysis carried out. Found

Chloroform-acetone 7: 3 ... Uniform Rf 0.4 were: lysine 0.96; Ammonia 0.28; Valine 2.0;

Benzene-acetone 1: 1 uniform Rf 0.62 proline 2.13; Tyrosine 0.87.

13 14

^Eg * ^{e 1 2} f) Lys (Pht) -Arg (NO ₂ ) -Arg (NO2) -Pro-Val-

a) BOC-ArgCNO ^ -Pro-Val-LysiPhQ-Val-Tyr-Pro- Lys (Pht) -Val-Tyr-Pro-OPAB

OPAR

^ ^Λβ 3.40 g of BOC nonapeptide ester are, as in the case of

Described from 893 mg of BOC-ArgiNO ^ -OH and 2.05 g of Pro- 5 game 1, e), treated with trifluoroacetic acid.

Val-Lys (Pht) -Val-Tyr-Pro-OPAB are according to the yield: 3.30 g LyS (PM) -ATg (NO ₂ ) -Arg (NO ₂ ) -

method described in Example 1, d) 2.21 g = 83% Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB = 100% of the

obtained according to the theory of BOC heptapeptide. Theory.

Thin-layer chromatogram: io thin-layer chromatogram:

Chloroform-methanol 9: 1 main component 0.58 Chloroform-methanol 9: 1 Uniform Rf 0.12

Impurity 0.79 Dioxane — Water 9: 1 ........ Uniform Rf 0.62

The contamination can be removed more easily after Ab- g) BOC-Lys (Pht) -Lys (Pht) -Arg (NO ₂ ) -Arg (NO ₂ ) -

Separate cleavage of the BOC group, which is why the i ₅ Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB

Product is not cleaned at this stage. From 1355 mg BOC-Lys (Pht) -OH and 3.30 g

Lys (Pht) -ATg (NO ₂ ) -ATg (NO ₂ ) -Pro -Val-Lys (Pht) -

b) Arg (NO2) -Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB Val-Tyr-Pro-OPAB are analogous to those in Example 1, d) 3.06 g of the mixture described under a) are described method 3.43 g of BOC decapeptide ester

obtained according to the method described in example 1, e) with ^a ° ^{= 86} / ° ^{der Theone}

Trifluoroacetic acid reacted and worked up. The thin-layer chromatogram:

The raw product held is 30 times the amount of aluminum. " _Λ1 . , _{ιη Λ} ". , ..,. ,,,. _{n en}

miniumoxide (activity III) chromatographed. With chloroform-methanol 9: 1 unit, Rf 0.57

Chloroform-acetone 7: 3 can be the impurity _% dioxane-water 9: 1 uniform Rf 0.74

elute. The Arg (NO ₂ ) -Pro-Val-Lys (Pht) -Val- ²⁵ J ₁ ) Lys (Pht) -Lys (Pht) -Arg (NO ^ Arg (NO.s) -

Tyr-Pro-OPAB remains at the top of the column. Es Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB

is after ejecting the column with tetrahydro-. , "_. _ ^ ₁ ..,. . ,,. ".__

furan water 9: 1 extracted. Yield: 2.14 g = 76% ^{From the} _u BOC decapeptide ester, the BOC

j Tt ₁ P ₀₁ -Jp group according to the method described in Example 1, e)

30 split off method. 3.10 g of Lys (Pht) -Lys- are obtained

Thin-layer chromatogram: (Pht) -Arg (NO2) -Arg (NO2) -Pro-Val-Lys (Pht) -Val-

Chloroform-methanol9: 1 unit Hch Rf 0.13 ^ ¹ * ^^ ⁼ T ^ ί ^ ^β "-« tv- u- u *

The connection is uniform according to thin-film

ciBOC-ArgiNO ^ -ArgtNO ^ -Pro-Val-LysiPht) - chromatognuiim in chloroform-methanol (9: 1);

Val-Tyr-Pro-OPAB ³⁵ ^ = ⁰ ' ¹⁰ '

From 910 mg BOC-Arg (NO ₂ ) -OH and above 2.14 g $ Lys (Pht) -Lys (Pht) -Arg-Arg-Pro-Val-Lys (Pht) -

ArgCNO ^ -Pro-Val-LysiPhti-Val-Tyr-Pro-OPAB val-iyr-iTo-Uü

are according to the Me- 1.00 g Lys (Pht) - LyS (PhI) -ATg (NO ₂ ) -ATg (NO ₂ ) - described in Example 1, d)

method 2.33 g = 87% of theory BOC octapeptide 4o Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB, dissolved in

obtained ester. 150 ml of 90% acetic acid, in the presence of

_. ,. 1., 200 mg of 10% palladium carbon for 20 hours

Thin-layer chromatogram: _{hydrogenated at 5 atmospheres water} pressure. After sucking off

Chloroform — methanol 9: 1 uniform Rf 0.36 of the catalyst becomes the solvent in vacuo

^ λ _Λ τ _Λΐ λ _n . _m ^ τ. τγ! τ / ™ χ ⁴⁵ completely evaporated. The residue is

d) ArgiNO ^ -ArgiNO ^ -Pro-Val-LysiPht) - _rub ^ _{water firmly after} sucking off and washing

Val-Tyr-Pro-OPAB ^ _{water and} 2n-acetic acid remain 750 mg Lys (Pht) -

From 3.4 g of BOC octapeptide ester, according to the Lys (Pht) -Arg-Arg-Pro-VaI-Lys (Pht) -VaI-Tyr-

Example 1, e) method described 3.3 g ATg (NO ₂ ) - Pro-OH, 2 CH ₃ COOH = 82% of theory back.

Arg (NO; j) -Pro-Val-Lys (Pht) -Val-Tyr-Pro-OPAB 5 <> The UV spectrum, recorded in 90% acetic acid,

= 100% of theory received. shows that there is no more nitroarginine present.

Thin-layer chromatogram: k) Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-OH,

Chloroform-Methanol 9: 1 Uniform Rf 0.03 ^{2 CH} sCOOH

^ τ, ηη τ / dua λ ^ t ^. λ λ / tvt ^ λ τ> ^{55 250 m} § Lys (Pht) -Lys (Pht) -Arg-Arg-Pro-Val-Lys-

e) BOC-Lys ^ -ArgJNO ^ -ArgiNO ^ -Pro- (Pht) -Val-Tyr-Pro-OH, 2 CH ₃ COOH, will

Val-Lys (Pht) -Val-Tyr-Pro-OPAB \ _{A s} ^ _nden ^ _{t 25 M of a meth} ^ _o ii _{scllen solution} ,

1.29 g BOC-Lys (Pht) -OH and 3.1 g octapeptide- which is 2 molar of hydrazine monoacetate (pH 6.5)

As described in Example 1, d), esters are stored at around 50 °. After concentration of the solvent

puts. The work-up is a little different: the ^{aus- 6o} is briefly heated with acetone, then the peptide with

Fallen triethylammonium chloride is filtered off with suction, ether precipitated. By dissolving in water, little can be achieved

and the filtrate dripped into ether. The precipitate will separate incompletely cleaved peptide;

sucked off. Yield: 3.56 g of BOC nonapeptide ester from the clear solution, the peptide is purified by lyophili-

= 92% of theory. sieren obtained as a white, hygroscopic powder.

_ ..,. ,. , 65 After total hydrolysis, a quantitative amino

Thin-layer chromatogram: acid analysis carried out: found = lysine 2.64;

Chloroform-methanol 9: 1 uniform Rf 0.5 arginine 1.70; Ammonia 0.84; Proline 1.88; Valine 2.00;

Dioxane-Water 9: 1 Uniform Rf 0.73 Tyrosine 0.86.

15 16

..-, ■■■; .. Example 3 formamide was added dropwise. After 1 hour at -5 ° and

15 hours at 0 ° is carried out in the usual way on new

a) BOC-Lys (Pht) pentachlorophenyl ester tral products worked up (in ethyl acetate). The product

wkdausätherkristamsiert ^ TSgBOC-Pro-Val-NHiä,

■ 50.9 g BOC-Lys (Pht) -OH and 43.2 g pentachloro 5 F. 85 ° (decomposition),

phenol are dissolved in 160 ml of absolute ethyl acetate / -inrm

And 30.7 g of dicyclohexylcarbodiimide were added at 0 °. ^d -> «o-Val-WH. ,, CM ₃ CUUM

After standing overnight at 0 °, the dicyclohexyl-7.73 g of BOC-PrO-VaI-NH ₂ becomes with cooling

Suck off urea and dissolve it well with ice-cold vinegar in 25 ml of triforoacetic acid. The solution will be

ester washed. The filtrate is evaporated in vacuo to io after 5 minutes in vacuo and the back

Dry evaporated and the residue from 500 ml was crystallized from ethanol-ether. Yield: 7.5g

Recrystallized ethanol. A first fraction of PrO-VaI-NH ₂ , CF ₃ COOH is obtained; F. 167 to 168 ° (decomposition

of 40.8 g of pentachlorophenyl ester with a melting point of 140 ° to 142 °. setting).

Is obtained from the mother liquor, a second fraction 7.0 g of the salt in 50 ⁰ / _O igrai methanol

obtained from 16.7 g, total 68 ° / ₀ of theory. ig solved and. by a column of a strongly basic one

. For the analysis is again recrystallized anion exchanger from ethanol, z. B. of the under the brand

siert, whereupon the melting point is 142 to 143 °. Amberlite IRA-400 is a well-known product from

· ■ ■ - "" · .-..- .. Rohm and Haas (in "the acetate form) run.

"· B) BOC-LysiPhQ-ArgCNO ^ -ArgiNO ^ -Pro- The filtrate is evaporated in vacuo and the return

VaI-T WPhtWai-Tvr-Pro-OPAB ^ao stood out from ether — petroleum ether crystallized; 5.66 g

vdtbywvdiiyrno Pro-Val-NH 2l CH ₃ COOH, _{mp 137-138 °.}
1.06 g Arg (NO2) -Arg (NO ₂ ) -Pro-Val-Lys (Pht) -

Val-Tyr-Pro-OPAB, 850 mg BÖC-Lys (Pht) -Penta-. _e ) BOC-Lys (Pht) -Pro-Val-NH ₂
chlorophenyl ester and 2.5 ml of dimethylformamide

stirred for 17 hours at 40 °. After dilution 25 from 5.26 g of BOC-Lys (Pht) -OH and 1.67 ml of chlorine with chloroform, the protected nonapeptide ester isobutyl isobutyl ester in 30 ml of tetrahydrate is precipitated with ether in 30 ml, suction filtered and thoroughly furan with ether and 1, 94 ml of triethylamine washed at -10 °. Yield: 1.24 g = 98% of theory. The mixed anhydride is produced, which (without being isolated to product is identical to that obtained in Example 2, e)) with the solution of 2.73 g of PrO-VaI-NH ₂ , and like this can be converted into the free one Peptide can be transferred to 30 CH ₃ COOH in 14 ml of absolute dimethylformamide. ' ^! . is implemented. After · 15 hours of reaction time • ■. . . . (to 0 °) the solution is strongly concentrated in vacuo, ■ --- ■;. ·. ± 5 & i s ρ 1 e 1 4. ^ _He residue in Essigester and water and dissolved in • - ^': a) Z-Val-NH ^ ^he ^ usual manner at 0 ° (with dilute hydrochloric acid, ² "" 35 and sodium bicarbonate) up to neutral products

25, IgZ-VaI-OH (R. L. M. Sy η ge, Biochem. J., worked. The neutral product is made from boiling

42, p. 99 [1948]) are crystallized in 200 ml of absolute tetra-ethyl acetate; BOC-Lys (Pht) -Pro-Val-NH ₂ ,

dissolved hydrofuran and mixed with 13J8 ml of triethylamine. Yield 65%, mp 172.5 _: to 173 °. Thin-layer chrome

seitz-t. 'At -10', slowly stir and matographie on silica gel with the solvent

Fire resistance 13.25 ml of chloroformic acid-ethyl acetate-methanol 1: 3 shows that the compound

isobutyl ester added dropwise After the mixed is uniform (Rf = 0.73) and through the crystalline

Anhydride has formed (30 minutes), a small amount of impurity ^{is released at -10 seconds}

dry ammonia introduced (up to saturation). has been separated with Rf = 0.55. After a further i-5 hours at 0 ° the is formed

Z-VaI-NH ₂ suction filtered and recrystallized from ethanol 45. - f) Lys (Pht) -Pro-Val-NH ₂ HCl

siert-F. 205 to-206 ^o , yield almost quantitative (24 g). '"'."'""

- · - ■ - .Ig BOC-Lys (Pht) -Pro-Val-NH ₂ is in 10ml

"""'b) VaI-NH ₂ , CH ₃ COOH dissolved in absolute ethyl acetate (boiling), cooled quickly and, before crystallization begins, with 6 ml

20.3 g of Z-VaI-TStH ₂ are added to a mixture of 50 2.9N hydrochloric acid in ethyl acetate! After a few 160 ml of glacial acetic acid and 40 ml of water with the addition of time, the hydrochloride begins to crystallize. 2 g 10 ° / ₀ palladium carbon at atmospheric pressure hy- After 1 hour, the crystals are filtered off and until no more hydrogen consumed with driert. washed with absolute ethyl acetate and ether; 613 mg After filtration from the catalyst and evaporation of the (= 69% of theory). The filtrate can be obtained from the mother liquor and the residue is recovered from 20 ml of ethanol by another 98 mg (= 11%). Crystallized in paper; 13.4 g VaI-NH ₂ , CH ₃ COOH, m.p. 102 °. chromatogram shows the substance in

The acetate is mixed directly with the various systems as a uniform compound,
hydride from isobutyl chloroformate and tert-

Butoxy-carbonyl-L-proline implemented. _g ) BOC-Ser-Tyr-Ser-Met-Glu (OC ₄ H ₈ t.) - His-

⁶⁰ Phe-Arg-Try-Gly-Lys (Pht) -Pro-Val-NH ₂
c) BOC-PrO-VaI-NH ₂

504 mg BOC-Ser-Tyr-Ser-Met-Glu (OC ₄ H ₉ t.) - His-

From 13, OgBOC-PrO-OH, 8.75 ml of triethylamine and Phe-Arg-Try-Gly-OH (patent application C 24191

8.4 ml of isobutyl chloroformate will give the gel VIb / 12q), 208 mg of Lys (Pht) -Pro-Val-NH ₂ , HCl, and

mixed anhydride made (30 minutes, -10 °). 65 145 mg of dicyclohexylcarbodiimide are in 5 ml

The pyridine solution is then stirred for 5 days at 20 ° for the reaction mixture, with everything

11.15 g of VaI-NH ₂ , CH ₃ COOH in 50 ml of absolute goes into solution. The product comes with a mixture

Tetrahydrofuran and 30 ml of absolute dimethyl from absolute chloroform and methanol as jelly

failed. Excess tripeptide is removed by washing with water. The BOC-Ser-Tyr-Ser-Met-GluCCOiHstO-His-Phe-Arg-Try-Gly-Lys (Pht) -Pro-Val-NH _{2 obtained} is pure, as the thin-layer chromatogram on Al ₂ O ₃ shows

h) H-Ser-Tyr-Ser-Met-Glu-His-Phe-Axg-Try-Gly-Lys (Pht) -Pro-Val-NH ₂ , 3 F ₃ CCOOH

530 mg of the tridecapeptide derivative are dissolved in 7 ml of trifluoroacetic acid, kept at room temperature for 15 minutes and freed from the solvent in vacuo at a bath temperature of 40 °. Yield 518 mg of H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys (Pht) -Pro-Val-NH ₂ , 3 F ₃ CCOOH. It is electrophoretically pure; 1 spot at 13.2 cm (positive reaction with Ninhydrin, Sakaguchi, Pauly, Ehrlich) after 5 hours and 7 volts / cm in ln-acetic acid on paper.

i) CHgCO-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys (Pht) -Pro-Val-NH ₂

The compound obtained according to h), the Ser ¹ - «-amino group of which is free and the Lys ¹¹ -e-amino group of which is protected against it, is selectively acetylated on the α-amino group by means of the p-nitrophenyl acetate.

500 mg of the N ^e -phthalyl-tridekapeptidamid-tri-trifluoroacetate are treated in 3 ml of absolute pyridine and 0.4 ml of absolute dimethylformamide with 35 mm ^{3 of} triethylamine and 92 mg of O-acetyl-p-nitrophenol for 22 hours at room temperature. The reaction product is then precipitated with ether; 440 mg (= 91% of theory) CHgCO-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys (Pht) -Pro-VaI-NH ₂ (Lys ⁿ -Ne-phthalyl- «-MSH). It is electrophoretically pure; 1 spot at 14 cm (positive reaction with the reagents according to Pauly, Sakaguchi and Ehrlich) after 7 hours and 7 vol / cm in ln-acetic acid on paper.

k) Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-NH ₂ , CH ₃ COOH

440 mg of the Lys ¹¹ -N ^e -phthalyl-oc-MSH are heated to 50 ° for 15 hours in 20 ml of 2 molar hydrazine acetate solution in methanol. The solution is then strongly concentrated in vacuo, taken up in 20 ml of warm acetone (35 ° to 40 °) and the «-MSH is precipitated by slowly adding ether.

ίο The Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-NH ₂ , CH ₃ COOH («-MSH) separate from about 30% unreacted starting material in pure form (control by electrophoresis in pyridine-glacial acetic acid-water 9: 1:90, pH 5.9). The starting material can be completely converted into the desired «-MSH by reacting it again with hydrazine acetate.

Claims (3)

Patent claims: 1. A process for the preparation of lower κ, ω-Όϊ- amino-alkanoic acids containing peptides with simultaneous protection of the α- and co-amino group, characterized in that the ω-amino group of the «, co-diamino-alkanoic acid by the phthalyl radical and the α-amino group is protected by the tert-butyloxycarbonyl radical and that the tert-butyloxycarbonyl group is removed in a known manner by acidic agents from the peptides obtained by condensation in a known manner and, at the end of the synthesis, the phthalyl group is split off in the presence of hydrazine in a weakly acidic medium . 2. The method according to claim 1, characterized in that the splitting off of the phthalyl radical at a pH between 4 and 7. 3. The method according to claim 1 and 2, characterized in that for splitting off the Phthalyl radical hydrazine monoacetate is used. 609 539/415 3.66 © Bundesdruckerei Berlin