DE1917690B2 - PROCESS FOR THE MANUFACTURING OF PEPTIDES - Google Patents

Description

R - C - N

or R - C - N

RCOOH means N-protected amino acids and N-protected peptides.

Dk ¹ Se new activated connections reaser. with primary and secondary amines extremely long! '. At 7 _ phe - VaI - OCH ₃ from Z - Phe - OBT

uiK H. Vaj - OCH ₃ · HCl with the addition of 1 equi-VLv; -. t N-ethylmorpholine are already after? Minutes fco: ■■ '· C above 90 ° ₀ peptide is formed (-. Fi l \ I [Bei-5Γ- ^v - "φ · B ^e ' the N-HydroxN-uccimmide-tery 'de is only after 2 hours : OC reaches 90 ", -S :. '·. Another advantage is that the esters do not need to be isolated more, since 1-Hyör-v-benzotriazole with dicyclohev. Learbodiimide im G-vensatz to N-Hydroxysuccinimiu no Nebenprciiukte forms That can be par-ierchroma <ograph; s: ι h - .. -ehr easily on a in Chem VT (1964), ^pp:: 07..!... partially protected corticotn>::> - (ll-23) -tridecapeptide-amide with free \ -amino- £ r ..; Dimethylformamide or a similar solution. So after a few hours it is no longer detectable by chromium graph, but has reacted to form other compounds. Tridecapeptide still uiy.er changed even after 15 hours.

Have in the activation of N-protected peptides al-carboxyl terminal amino acid proline and no (il \ one has to reckon with a low racemization but can also be N-Hydroxvsuccinimidester ^m;. Dicycluhexy'carbodiimid not t Canz racer.iisierungsfrei produce (see Table 2 [bei · tpicll, 2]).

The compounds customary in peptide chemistry, such as N.N'-dicyclohexylcarbodiimide, can be used as carbodiimides. N.N'-Diisopropylcarbodiimide and water-soluble Carbodiimides can be used. As protecting groups for those functional groups of the amino acids and peptides that are to be protected, all protective groups customary in peptide chemistry are suitable. Also polymeric resins, such as. B. Hydroxymethylpolystyrene, can be used as protecting groups E. Schröder, K. Lübke, The Peptides. Vol. I, Academic Press New York and London [1965], pp. 108 to 111).

Suitable solvents commonly used in peptide chemistry are, for example, dimethylformamide, dimethyl acetamide, tetrahydrofuran, dioxane, pyridine, dimethyl sulfoxide or methylene chloride. The reaction temperature is advantageously between -20 and + 4O ³ C, preferably around O ⁰ C.

In the case of carboxyl-protected peptides which are sparingly soluble in water, the added 1-hydroxybenzotriazole can be used with sodium or potassium bicarbonate solution or shake completely with soda solution.

Fin is a particular advantage of this method. that he

by acidifying these sodium bicarbonate wash solutions the 1-hydroxybenzotriazole used can precipitate again. This is e.g. B. with an addition of N-hydroxysuccinimide not possible because N-Hy-

■ '5 droxysueeinimid also dissolves in acids. From poorly soluble One can peptides with isopropanol, alcohol, methanol. Tetrahydrofuran or hot water that Extract 1-H \ dr Avbenztriazole.

The prey was in all of the examples examined

-o with the addition of 1-hydroxybenzene: .zol higher than with or without the addition of N-hydroxysuccinimide. For example, in protected decapeptide with the sequence of the antamanids. a cyclodecapeptide from Aman ^; a phalloides from Z - Phe - Phe - Pro - Pro - OH and H - Phe - Phe - VaI - Pro - Pro - AIa - OBu 'according to the dicyclohexylcarbodiimide method with an addition of 1-hydroxybenztria? ol at 91.4 °. ₀ yield can be synthesized. A corresponding addition of N-hydroxysuccinimide yielded only 61 ⁿ ₀ .

The new process also proved itself in production \ on Corticotropin-) l-23) -trikosapepnd-amid analogous to the German patent 1,240,088 Addition to the dicyclohexyl-carbodiimide 4-nitrophenol used. Even better than this addition has proven itself but pentachlorophenol or hydroxysuccinimide. If one puts the achieved with these latter additives Yield - = 100. so one finds without addition; ■ 60. with 4-Nitrophenol = 80. with 1-Hydrox \ benzotriazole however - 140. as both the biological evaluation and a quantitative method of determination was based on the photometric evaluation of the color of the ninhydrin-cadmium complex analogous to one in natural science. 42 (1955), p. 416, described method after separation of the crude product by paper chromatography is based.

As Table 1 shows, the Recamisierung decreases in the dicyclohexylcarbodiimide method in tetrahydrofuran as a solvent to l ° _0th when 2 equivalents of 1-hydroxybenzotriazole are added. Used

we dimethylformamide as the solvent, 1-hydroxybenzotriazole occurs upon addition of 2 equivalents of a low racemization, while b "\ the addition of 1-1.2 equivalents of racemization below 1 ° '" remains. In addition, N-hydroxysuccinimide, it is just vice versa. If a peptide via dicyclohexylcarbodiimide and 1-hydroxybenzotriazole activated, the racemization is also here with 1 equivalent of 1-hydroxybenzotriazole at a minimum. In tetrahydrofuran they even remain below 1 ⁰ I _n. In the case of N-hydroxysuccinimide-addition is also here the gear again vice versa (see Table 2). As Examples III, 2 and 3 (synthesis of BUC-GlN [Bz] -OBz) show, the yields with the addition of 2 equivalents of N-hydroxysuccinimide are lower than with 1 equivalent, and the Reaction products are also more heavily contaminated, which means that when N-hydroxysuccinimide is added, the low level of racemization must be paid for with the formation of many by-products.

The new activated esters were isolated in individual cases. It is interesting that with Z-Thr-OH the 1-hydroxybenzotriazole ester was producible, while at Z - Ser - OH the / i-lactone of Z - Scr - OH was formed. In the one-pot process, however, Produce serine peptides in excellent yield. For example, Z - Ser-GIy - ONB could be added by the dicyclohexylcarbodiimide method from 2 equivalents of 1-hydroxybenzotriazole in 97.5% yield will. Without the addition of 1-hydroxy-benzotriazole the yield was only 70%, (R. S c h w y ζ e r, HeIv. Chim. Acta, 42 [1959], p. 1702).

This new process is therefore of great importance for the production of serine peptides, because the the azide method, which is mostly used, gives lower yields. Also arises from N-carbobenzoxy-serine-azide 4-carbobenzoxy-amine-oxazolidinone-2 via the isocyanate as a by-product. When dicyclohexylcarbodiimide is used alone, the result is By-product N-acylurea (E. Schröder and K. Lübke, The Peptides, Vol. I, Academic Press, New York and Londen, p. 208 [1965]).

If glutaminyl or asparaginyl peptides are produced using dicyclohexylcarbodiimide, this is how it is produced the corresponding NiUIe by dehydration of the acid amide group in a considerable amount. at Imide formation is also possible with N-acyl asparagine. Asparaginyl peptides can only be produced in yields of 39 to 45% (E. Schröder and K. Lübke, Peptides, Vol I, Academic Press, New York and London, pp. 191 and 202-204 [1965]). at Addition of 1-hydroxybenzotriazole to the dicyclohexylcarbodiimide method however, glutaminyl and asparaginyl peptides can be quickly obtained in good yields and present it cleanly. So was z. B.

Z - AsN - Leu - OCH ₃

according to the new method in 85% and
Z _ GIN - AIa - OBu «

AsN = asparagine

GIN = glutamine

Z = carbobenzoxy-

Boc = tert-butyloxycaibonyl-

NPS = o-nitrophenylsilfenyl-

OCH ₃ = methyl estci

ONB = p-nitrobenzyl ester

OBu ⁴ = tert-butyl ester

ONp = 4-nitrophenyl ester

ίο OSu = N-hydroxysuccinimide ester

OTCP = »trichlorophenyl ester

OBT = 1-Cxy-1 ^ .S-benzotriazole ester

Mbh = 4,4'-dimethoxy-benzhydryl-

Mmb = α a-dimethyl-4-methoxy-benzyl-

DMF = dimethylformamide

CHA = cyclohexylamine

DCHA = dicyclohexylamine

DCC = dicyclohexylcarbodiimide

TLC = thin layer chromatography

ao OBz = benzyl ester

THF = tetrahydrofuran

Bz = benzyl

DMA = dimethylacetamide

All of the following amino acids are used in their L-form.

Examples

1. Gas chromatographic racemization test according to F. Wegand et al.

(F. W ey g a η d, A.P 1 ο χ and W. K ö η i g, Chem. Ber., 99 [1966], pp. 1451 to 1460).

The test was modified so that BOC - Leu - Phe - OH was used instead of Z-Leu-Phe-OH which has the advantage that the resulting completely protected peptide BOC - Leu - l.d - Phe - VaI - OBu ' can be hydrolyzed without prior splitting off of protective groups.

received in 73.7%.

Also arginine peptides with protonated guanido groups can be produced in pure form and with a better yield using the new method. Even the Representation of the activated ester from protonized N-acyiarginine and 1-hydroxybenzotriazole, which without Isolation is implemented further is possible.

^{All A ”:} ao acids in their L or D form that are found in naturally occurring peptides can be used as building blocks of the peptides produced by the process according to the invention. The use of ^ amino acids, such as. B. / ^ - alanine or other only synthetically or semi-synthetically accessible amino acids, z. B. "-Methylalanine," -Methyl-3,4-dioxy-L-phenylalanine or / J-chloralanine is possible.

After the protective groups have been split off, the products of the process can be used as therapeutics find or as intermediates zui production of other therapeutically valuable peptides such. B. Oxytocin, vasopressin, glucagon, ACTH, secretin, thyreocalcitonin or insulin can be used.

In the description and in the examples, the amino acids are used according to the internationally valid ones Abbreviated rules The following abbreviations are also used:

Manufacture of BOC - Leu - Phe - OH

38 g of phenyla'anine are dissolved in 345 ml of dioxane and 115 ml of 2N sodium hydroxide solution. 38 g of BOC - Leu - OSu are added and the mixture is stirred for about 20 hours at room temperature. The precipitate (excess phenylalanine) is filtered off with suction and the filtrate is concentrated. The residue is distributed between 200 ml of ethyl acetate and 220 ml of 2η-citric acid. The ethyl acetate phase is extracted once more with 2N citric acid and water, dried with sodium sulfate and concentrated. The residue is triturated with petroleum ether. Yield 39.3 g, m.p. 108 to 111 ° C. Recrystallized from ethyl acetate-petroleum ether, yield 33.3 g, melting point 112 to 115 ° C., [<x) _D = -8.5 (c = 2, methanol).

Production of H-VaI- 0Bu ^{ · HQ

N-carbobenzoxy-L-valine tert-butyl ester was i: Methanol with Pd catalyst with the aid of an autotiitrator at pH 5 (addition of 1 N-me thanolischei HQ) catalytically hydrogenated.

H - Val - OBu'- HQ can be crystallized from Essigester vat. Mp. 148 ° C, [a] _D + 19.22 ° (c =: methanol).

a) Testing for racemization using the dicyclohexylcarbodiimide method with different

Additives

37?.; mp BOC - Leu - Phc - OI; (ImMoI) and 209.7 mg H - VaI - OBu '· HCl (ImMoI) are dissolved in 2 ml of absolute DMF or tetrahydrofuran. Add 0.12 ml of N-ethylmorpholine (1 mmol) and cool in an ice bath. The additives are then added and, finally, a solution, cooled to O'C, of 207 mg of DCC (1 mmol) in 1 ml of absolute dimethylformamide or tetrahydrofuran. The batches are left to stand for 5 hours at ⁰ ° C. and for about 15 hours at room temperature, diluted with about 30 ml of ethyl acetate, the precipitate is filtered off, the filtrate is shaken with saturated sodium bicarbonate solution, 2η-citric acid, saturated sodium bicarbonate solution and water, and dried with sodium sulfate, and the residue is chromatographed in ethyl acetate over 3 g of basic aluminum oxide (W ο elm, active level I). The eluate (approximately 40 ml) is concentrated and the residue is dissolved in about 5 ml of 8 to 9 N-methanolic hydrochloric acid and heated for 24 hours 7O ⁰ C in a sealed tube. The methanolic hydrochloric acid is concentrated and work continues as with F. Weygand et al.

The results are summarized in Table 1 below.

Table 1

Racemization studies in dei dicyclo-

hexylcarbodiimide method with various

Additives

Equi
valent Addition no solutions D-Phe-L-Val
^U / o 0 no DMF 14.3 0 Pentachlorophenol THF 8.1 2 Pentachlorophenol DMF 27.0 2 N-hydroxysuccinimide THF 10.0 1 N-hydroxysuccinimide DMF 1.0 1.2 N-hydroxysuccinimide DMF <l, 0 2 N-hydroxysuccinimide DMF <t, 0 2 l-hyoroxybenzotriazole THF <l, 0 1 1-hydroxybenzotriazole DMF <l, 0 1.2 1-hydroxybenzotriazole DMF <l, 0 2 1-hydroxybenzotriazole DMF U 2 THF <l, 0

b) Testing for racemization when activating

Peptides with dicyclohexylcarbodiimide and 1-hydroxy-

1,2,3-benzotriazole or N-hydroxysuccinimide

378.5 mg of BOC - Leu - Phe - OH (1 mmol) and additive are dissolved in 2 ml of absolute DMF or tetrahydrofuran. The mixture is cooled to ⁰ ° C., a chilled to 0 ⁰ C solution of 207 mg DCC (1 mmol) in a 1 ml of absolute DMF or tetrahydrofuran to iäßt and 2 hours at O ⁰ C standing. Then 209.7 mg of H-VaI-OBu '· HCl (1 mmol) and 0.12 ml of N-ethylmorpholine (1 mmol) are added, the mixture is left to ^{stand for a further 3 hours at 0} ° C. and set at room temperature for about 15 hours . Now it is worked up as in ΐ, 1

The results are summarized in Table 2 below.

Table 2

Rac »measurement examinations during activation of peptides with dicyclohexylcarbodiimide and 1-hydroxy-benzotriazole and N-hydroxysuccinimide, respectively

Equi
valent additive Solutions n-Phc-L-Val
'7o ¹⁰ ι
1.2
2
1
1 N-hydroxysuccinimide
N-hydroxysuccinimide
N-hydroxyrccinimide
1-hydroxybeiiztriazole
1-hydroxybenzotriazole
1-hydroxybenzotriazole
1-hydroxybenzotriazole DMF
DMF
DMF
DMF
DMF
DMF
THF 2.3
2.3
1.65
4.4
8.4
9.3
<l, 0

2. Synthesis of Z-VaI- VaI - OCH ₃ by the diao cyclohexylcarbodiimide method with various additives

a) Without addition

2.5 g of Z - VaI - OH (10 mmol), 1.7 g of H - VaI - OCH ₃ · a ₅ HCl (10 mmol) and 1.28 ml of N-ethylmorpholine (10 mmol) are dissolved in 20 ml of absolute tetrahydrofuran cooled to ⁰ ° C. with stirring. ^{A solution, cooled to 0} ° C., of 2.2 g of DCC in absolute tetrahydrofuran is added and the mixture is stirred for 1 hour at room temperature. The precipitate is filtered off with suction, the filtrate is concentrated, the residue is dissolved in ethyl acetate and the solution is washed with saturated sodium bicarbonate solution, 2η hydrochloric acid, saturated sodium carbonate solution and water, dried with sodium sulfate, concentrated and triturated with petroleum ether. Yield 70%, m.p. 99-107 ° C.

b) Addition of 2 equivalents of 1-hydroxy-benzotriazole

Approach as in 2, a). Before the addition of DCC, 2.7 g of 1-hydroxy-benzotriazole (20 mmol) are added. Yield 93.4%. Mp. 99 to 102 ⁰ C.

c) Addition of 1 equivalent of 1-hydroxy-benzotriazole

Approach as in 2. a). Before the DCC addition, 1.35 g of 1-hydroxy-benzotriazole (10 mmMol) are added. Yield 82.4%, m.p. 92-103 ° C.

d) Addition of 2 equivalents of N-hydroxysuccinimide

Approach as in 2, a). Before the addition of DCC, 2.3 g of N-hydroxysuccinimide (20 mmol) are added. Yield 68.7%, m.p. 76-82 ° C.

3. Synthesis of Z - VaI - VaI - OCH ₃ over

activated esters

a) With not insulated Z-VaI-OBT

2.5 g of Z - Val - OH (10 mmol) and 2.7 g hydroxy-1,2,3-benzotriazole (20 mmol) are dissolved in 20 ml absolute tetrahydrofuran and, at 0 ⁰ C with a cold solution of 2 2 g of dicycloherylcarbodiimide in absolute tetrahydrofuran are added. It is stirred for 1 hour 'at 0 ⁰ C and adds to stand for 1 hour at room temperature 1.7 g H-Val-OCH ₃ · HCl (10 mmol) unc 1.28 ml of N-ethylmorpholine (10 mmol) is added and let 1 hour at Stir at room temperature. Work-up as in 2. a). Yield 90.7%, mp. 103 to 107 ⁰ C.

309525/52

4-

b) With non-isolated Z-VaI-OSu (with 2 equivalents N-hydroxysuccinimide)

Approach as in 3, a). Instead of 1-hydroxy-benzotriazole, 2.3 g of N-hyuroxysiiccinimide (20 mmol) are added. Yield 72.8 °, ₀ , m.p. 74 to 85 C.

c) With nich. isolated /.-VaI-OSu (with 1.1 equivalents N-hydroxysuccinimide)

Approach as in 3, a). Instead of 1-hydroxy-benzotriazole, 1.25 g of N-hydroxysuccinimide (11 mmol) are added. Yield 81 ° / ₀ , m.p. 82 to 86 = C.

d) With isolated Z - VaI - OSu

3.5 gZ-VaI -OSu (10 mmol), 1.7 g H-VaI-OCH ₃ · HCl (10 mmol) and 1.28 ml N-ethylmorpholine (10 mmol) are stirred in 20 ml absolute tetrahydrofuran for 2 hours at room temperature and worked up as in 2, a). Yield 82.9%, m.p. 94 to 97 C.

4. Synthesis of BOC - GlN (Bz) - OBz a) With non-isolated BOC - GIu (OBT) OBz

To a solution, cooled to OX, of 3.4 g of BOC-GIu-OBz (10 mmol) and 1.5 g of 1-hydroxybenzotriazole (11 mmol) in 20 ml of absolute tetrahydrofuran is added a solution of 2, also cooled to O "C, 2 g of DCC in absolute tetrahydrofuran are allowed to stand for 1 hour at 0 ° C. and 1 hour at room temperature, then 1.1 ml of benzylamine are added and the mixture is left to stand for a further hour at room temperature The residue is dissolved in ethyl acetate and the solution is extracted successively with saturated sodium bicarbonate solution, 2η-citric acid, saturated sodium bicarbonate solution and water, dried with sodium sulfate and concentrated. The residue is triturated with petroleum ether. Yield 3.6 g (84.4%), melting point. 87 to 88 ° C. For further purification, the substance is dissolved in ethyl acetate and chromatographed over about 20 g of basic aluminum oxide (Wo elm, active level I.) The eluate is concentrated and the residue is triturated with petroleum ether. Yield 2.9 g (68.1%). The substance is chromatographically pure. M.p. 92 to 94 ^r C [*] d -19.9 (c = 2, methanol).

b) With non-isolated BOC-GIu (OSu) -OBz (with

2 equivalents of N-hydroxysuccinimide)

Approach as in 4, a). Instead of 1-hydroxybenzotriazole 2.3 g of N-hydroxysuccinimide (20 mmol) are added. The residue was not with petroleum ether crystallizable. After purification on a basic aluminum hydroxide column, 1.75 g (41.1%) crystallized. M.p. 75 to 77 ° C. The substance was chromatographic not in.

c) With non-isolated BOC-GIu (OSu) OBz (with

1 equivalent of N-hydroxysuccinimide)

Approach as in 4, a). Instead of 1-hydroxy-benztridzene, 1.15 g of N-hydroxysuccinimide (10 mmol) are added. The residue could not be crystallized with petroleum ether. After purification on a basic aluminum oxide column, 2.2 g (51.5%), melting point 88 to 90 ^° C., crystallized. The substance was almost clean according to thin-layer chromatography.

5. Peptide syntheses with Z - Phe - OBT ρ) Ζ-Phe-OBT

To a solution cooled to OC 30 c Z-Phe-OH (0.1 mol) and 15 g of 1-hydroxy-benzotriazole (0.11 mol) in 300 ml of absolute tetrahydrofuran is added a solution which has also been cooled to O'C of 22 g of DCC. The mixture is left to stand at OC for 1 hour and at room temperature for 1 hour, the precipitate is filtered off with suction and the filtrate is concentrated. The residue is recrystallized from isopropanol. Yield 28.9 g (69.5%), m.p. 120 to 122 ^° C.

b) Z - Phe - Phe - OCH ₃ 1.2 g of H - Phe - OCH ₃ · HC) (5 mmol) are suspended in 20 ml of absolute tetrahydrofuran. The mixture is cooled to 0 ° C., 0.7 ml of N-ethylmorpholine (5 mmol) is added and the mixture is stirred for 5 minutes. Then 2.1 g of Z - Phe - OBT are added and the mixture is stirred overnight. Change-

the day the precipitate is filtered off with suction, the filtrate is concentrated, the residue is dissolved in ethyl acetate and worked up as in II.1. Yield 2.15 g (91%), melting point 148 to 150 ° C., h] D -17.95 ° (c = 2, in dimethylformamide).

* ⁵ c) Z - Phe - VaI - OCH ₃

0.9 g of H-VaI-OCH ₃ (5 mmol), 0.7 ml of N-ethylmorpholine and 2, IgZ-Phe - OBT are reacted as in 5, b). Yield 1.95 g (94.5%), mp 10-112; c.

Chromatographed over 20g of basic aluminum oxide (Woclm, active level I) in ethyl acetate: Yield 1.7 g (82.5%), melting point 111 to 113'C [*] n -8.9 ' (c = 2, in dimethylformamide) .

d) Kinetic studies on the synthesis of Z - Phe - VaI - OCH ₃

115.2 mg of N-ethylmorpholine (ImMoI) are dissolved in 5 ml of absolute tetrahydrofuran cooled to ^{0 ° C.} To this are added 184 mg of H - VaI - OCH ₃ · HCl (1.1 mmol) and finally, with stirring, 458 mg of Z - Phe - OBT (1.1 mmol) or 436 mg of Z-Phe-OSu (1.1 mmol). The ^{mixture is then stirred at 0} ° C. The reaction is stopped at the desired point in time by adding 10 ml of 0.1 η hydrochloric acid. With 0.1 N sodium hydroxide solution _ir w d is the free hydrochloric acid potentiometrically using a glass electrode titrated. The amount of 0.1 N sodium hydroxide solution consumed is proportional to the yield of peptide. In Fig. 1 shows the results of this kinetic investigation.

6. Synthesis of a protected decapeptide with the antamanide sequence (Z - Phe - Phe - Pro - Pro - Phe Phe - VaI - Pro - Pro - Ala - OBu *)

a) Z - Pro - Pro - OH (MW. 346) 37OgZ- Pro - ONP (1 mol) and 127 g (1.1 mol) Pro are in the presence of 140 ecm (1 mol * tri-

ethylamine boiled in ethanol for 4 hours. Man the solvent is removed in vacuo, the residue is taken up in 1.5 l of water and extracted twice each with 250 ecm ether. The aqueous phase is then acidified to pH 2 with half-concentrated HCl

an and maintains a crystalline precipitate, which is recrystallized from a little ethanol. Yield 256 g (74%) melting point 188 to 18> ° C [x]% -10Γ (c = 2 in 50% DMF).

b) H - Pro Pro-OH (MG 212)

60 g (0.173 mol) Z-Pro-Pro-OH are hydrogenated on Pd in 500 ecm SO percent methanol. After the catalyst has been filtered off, the solvent is distilled off in vacuo, the residue is triturated with acetone, filtered off and washed with ether. Yield 35.2 g (96 ° / ₀ of theory) [x]% ° "- 160 ^r (c = 2 in water).

c) Z - Phe - Phe - OH (MG 446)

66 g of Phe (0.4 mol) are dissolved in a mixture of 800 ecm dioxane and 200 ecm 2 N NaOH. 79.2 g (0.2 mol) of Z - Phe - OSu are added and the mixture is stirred at room temperature overnight. Now the precipitated Phe is sucked out. The filtrate is evaporated to dryness in vacuo, the residue is taken up in 400 ecm of ethyl acetate, extracted with 2N HCl and water, the ethyl acetate phase is dried over sodium sulate and the solvent is distilled off. The residue is recrystallized from ethanol-water 3: 1. Yield 69.0 g (77.4% of theory) Melting point 158 to 16O ⁰ C, molecular weight calculated 446. found (titration) 445th

d) Z - Phe - Phe - Pro - Pro - OH

From 44.6 g (0.1 mol) of Z - Phe - Phe - OH, 23 g (0.2 mol) of HOSu and 22 g (0.107 mol) of DCC, in 400 cm of THF in the usual way at -5 C of the hydroxysuccinimide ester manufactured. After the addition, the mixture is stirred

5 "C and 2 hours

at room temperature, sucks off the urea (2.3 g) and distills the mixed solution in vacuo. The residue is recrystallized from ethanol. Yield 42.5 g (78.2 ° / ₀ ), melting point 148 to 151 ⁰ C [λ] Ό ' -30.9 ° (c = 1 in DMA). 37.8 g (70 mmol) of this hydroxysuccinimide ester are stirred together with 15.4 g (73.5 mmol) of H-Pro-Pro-OH in 150 ecm DMF. After 45 minutes, all of the H - Pro - Pro - OH has gone into solution. 8.1 g (70 mmol) of N-ethylmorpholine are added and stirring is continued overnight at room temperature. After the solvent has been distilled off in vacuo, the residue is taken up in 200 ecm of ethyl acetate, extracted with 1N HCl and a little water, dried over sodium sulfate and the ethyl acetate is distilled off. The residue is taken up in a little acetone. Upon addition of 8.5 ecm of cyclohexylamine and 300 ecm of ether, a precipitate separates out, which, after standing for several hours at ⁰ ° C., is filtered, dried and re-crystallized from isopropanol. Yield 29.0 g (64.8%) melting point 194 to 196 ° C [*] £ -101 ° (c = 0.3 in methanol) C ₄₂ H ₅₂ N ₅ O ₇ (739.8),

Calculated ... C 68.2, H 7.08, N 9.47%,
Found ... C 68.2, H 7.2, N 9.6%.

To release the acid, the compound is taken up in ethyl acetate and shaken twice with each 50 ecm 1 N-HCl, washed with a little water, dried over sodium sulfate and distilled the ethyl acetate in the Vacuum off. Almost quantitative yield.

e) Synthesis of H - Pro - Pro - AIa - OBu '· HCl

aa) According to the dicyclohexylcarbodiimide method with the addition of 2 equivalents of 1-hydroxy-benzotriazole

To a solution of 3.46 g of Z - Pro - Pro - OH (10 mmol), 1.82 g of H - AIa - OBu '· HCl (10 mmol), 2.7 g of 1-hydroxy-benzotriazole (2OmMoI) and 1.28 ml N-ethylmorpholine (10 mmol) in 30 ml of absolute DMF is added to a cold solution of 2.2 g of DCC in absolute DMF at OC. The mixture is left to stand for 2 hours at O ^: C and 1 hour at room temperature, the precipitate is filtered off with suction, concentrated and the residue is partitioned between ethyl acetate and water. The ethyl acetate phase is successively with saturated sodium bicarbonate solution, 2 η-citric acid, saturated; Washed sodium bicarbonate solution and water, dried with sodium sulfate and concentrated. The residue is chromatographed over about 20 g of basic aluminum oxide (W ο el m, active level I) in ethyl acetate. The eluate is concentrated. Yield of oily Z - Pro - Pro - Ala - OBu '4.0 g (84.5%).

The oily Z - Pro - Pro - AIa - OBu 'is in methanol catalytically hydrogenated with the addition of a palladium catalyst. With the help of an autotitrator, Addition of 1 N-methanolic hydrochloric acid to a pH of 5

ao complied with. After the reaction has ended (no further Uptake of methanolic hydrochloric acid), the catalyst is removed, the filtrate is concentrated and mixed with Ether rubbed in. Yield 2.75 g (73.2%, based on Z - Pro - Pro - OH), the substance is chromato-

»5 graphically uniform. Mp. 147-151 ⁰ C [\] r> -152 '(c = 2 in methanol).

bb) According to the dicyclohexylcarbodiimide method with the addition of 2 equivalents of N-hydroxysuccinimide

Approach as in 6, e), aa), instead of 1-hydroxy-benztriazo! 2.3 g of N-hydroxysuccinimide (20 mmol) are added. Yield of oily Z - Pro - Pro - OBu ^c 3.1 g (65.5 o / o).

After the catalytic hydrogenation, 1.8 g of H - Pro - Pro - Ala - OBu '· HCl can be isolated (47.8%, based on Z - Pro - Pro - OH). Mp 147-151 ⁰ C. thin layer chromatography. Could be detected in lanes a ninhydrinnegative substance.

cc) With non-isolated Z - Pro - Pro - OBT

^{A solution of is added at 0} ° C. to a solution of 3.46 g of Z - Pro - Pro (10 mmol) and 1.5 g of 1-hydroxybenzotriazole (11 mmol) in 30 ml of absolute DMF

«2.2 g DCC, dissolved in cold DMF. Leave for 1 hour stand at O'C and 1 hour at room temperature and then add 1.82 g of H - AIa - OBu '· HCl (10 rr "* ol] and 1.28 ml of N-ethylmorpholine (10 mmol). After another hour at room temperature, like at 6, e), aa) worked up. 4.45 g of oily Z - Pro - Pro - Ala - OBu '(94.1%) are obtained.

After the catalytic hydrogenation, 3.12 § H - Pro - Pro - Ala - OBu '· HCl are obtained (83% be attracted to Z - Pro - Pro - OH), m.p. 145 to 150 ° C. The Substance is chromatographically uniform.

f) Z - VaI - Pro - Pro - AIa - OBu "

To a solution of 4.6 g of Z - VAI OH (18.3 mmol and 2.75 g of 1-hydroxy-benzotriazole (20.4 mmol) ir 45 ml of absolute tetrahydrofuran are added at 0 ⁰ C, a cold solution of 4 .05 g of DCC in absolute tetrahydrofuran. The mixture is left to stand for 1 hour at ⁰ ° C. and for 1 hour at room temperature and 5.8 g of H-Pro-Pro-Ala-OBu * · HCl (15.5 mol

and 1.98 ml of N-ethylmorpholine (15.5 mmol). Mai stirs 1 hour at room temperature, sucks Precipitate from, the filtrate is concentrated and worked up wi at 6, e), aa). Yield 8.4 g of oil (95%).

⁰ I.

"(Ζ- Phe - Phe - \ al - Pro - Pro - Ala - OBu ^! The residue is dissolved in 40 ml of DMF and mixed with

7.3 er H - Phe - Phe - YaI - Pro - Pro - Ala - OBu '

S.4oZ- YaI - Pro - Pro - AIa - OBu ^! (14.67mMol) (10 ϊηΜοΙ) and 2.7 g 1-Hydroxy-benztriazoi (2U mmol)

are added in methanol with the addition of palladium. The mixture is cooled to -10 ° C. and a solution is added

catalytically hydrogenated. With the help of an auto 5 \ on 2.2 g DCC. dissolved in cold DMF. to that, lets

titrators by adding 1 n-methanol for 4 hours at low temperature and overnight at

Maintain hydrochloric acid at pH 5. After standing at room temperature, the next day -in narrow. takes

Reaction, the catalyst is filtered off. the Ekiat on the residue in ethyl acetate and shakes as in

concentrated and evaporate the residue with ether. 6. egg. eel out. The back wall is made in tetrahydrofuran

The ether is decanted off and the oil is dissolved in high io and over a basic aluminum oxide column

vacuum dried. The result is an amorphous mass. Chromatographs. The eluate is concentrated and im

of 6.8 g U4.3 mmol) H -YaI -Pro- Pro -AIa- OBu '· dried under high vacuum. It remains a .ünorph.

HCl (97-6 ^fl "). aie together with 6 4 ^ a thin-film chromatographically uniform sur- tar./

Z-Phe-Phe-OH * _ back. Yield 12.4 g (91.4 _% ).

... .. ,, .., ",, - ,,,, -.,. ^a bb) With the addition of 2 equivalents of NH \ oroxv-

(14.4 mmol. 3, see: ■ gi-hyuroxy-benztnazoleCfomMol) s lcc'inimide and 1> 3 ml of N-ethylmorpholine (14.3 mmol) in 50 ml

absolute ·, DMh is solved. With OC one gives an approach as in 6. i). aa). Jam 1-Hydroxy-K : -

cold solution of 3.15 g DCC in absolute DMF. triazole become 2 equivalents of N-Hydro \\? - jv-c :: i: r.v ^.

KiBt added for 2 hours at 0 C and 1 hour at room temperature. Yield 61 ° _y

stir temperature, sucks off the precipitate. narrows the ~ AsN (MbM - Cvs (Mmb) - Pro Lv, -, Roe Filtrate. takes up the residue in ethyl acetate "Cl - * NH-Mbh and shakes out as in 6th el. aa). dries with sodium sulfate, constricts and rubbed with petroleum ether. al H GIy - NH - Mbh · HCl

Yield 11.2 c (88 ⁿ _0. Be / .oaen on inserted 25..., -, _vu »,,,

-7 λ- ι η r>*>, λγ, ";> τ r. · ■" 1 aa ¹ Aus / - CiIv - NH Mbh

Z - \ al - Pro - Pro - AIa - OBu '). Used for cleaning

in tetrahydrofuran over basic aluminum oxide 44. "g Z-GK-NH-Mbh are in an M-

(W ο elm. Active level 1) Chromatographies. Yield loop from 3 GO ml of methanol and 300 ml of ice cream - ·. -

9.05 g (71.1 "" based on pendulum and catalysis with palladium catalyst.

30 hydrogenated. The catalyst is suctioned off mw. ■, '.. ·. ·

Z-VaI-Pro-Pro-AIl -OBu ') ρ _{Πιπ1ι emgee} ngt. The residue is ii. Meth ..: v

The melting point is not sharp (10 to 145 C). dissolved and against thymol blue with methanolic>; .'.-

[\] r> -126 (c 2nd in methanol). The substance is titrated with acid. Now it is narrowed down again vvJ , ::; ·

chromatographically uniform. Triturated residue with ether. Yield. "·. · .'T;

Instead of 1-hydroxy-benzotriazole, 2 equivalents 35 1.98 ° "). Recrystallized from methane! Ether:. ·;. "" ■ -

Added N-hydroxysuccinimide, 7b ' ₍₁ crude (9i.6 " _Λ ). Mp. 202-204 C. Z - Phe - Phe - VaI - Pro'- Pro - AIa - OBu' isolated.

From this raw substance after cleaning bb) from chloressic acid

over aluminum oxide only about 75 " _n again 4,4'-dimethoxybenzhydrylamide

be won. [\] u 126 U - 2. in methanol). 40

, ,, ",",, To a solution of S8.5 s chlorine> vi; s; iure-4.4'-o: -

h) H - Phe - Phe - \ al - Pro - Pro - AIa - OBu 'methoxybenzhydrylamia Ir. 880 ml of methanol are added

To a solution of 8.9 g 220 ml condensed. \ Mmonia ^ and heated in the Arno-

Z- Phe - Phe - Pm - Pro - VaI - Pro - Pro - AIa - OBu ' ^{kUlV 24 hours} f " ^ai ' ^f .« ° ^bis ] ^{Q C} 'Then we w iro

45 concentrated and the residue between hssigoster uiu:

Palladium catalyst and soda solution are distributed in methanol. The diester phase is mixed with it as usual, with a 1N water wash being added dropwise. dried with sodium sulfate and concentrated ethanolic hydrochloric acid using an autoturator. The residue is dissolved in methanol and pH 5 is maintained. After the hydrogenation has ended, methanolic hydrochloric acid is added until the catalyst is removed and the filtrate has a pH of 6. It is now concentrated again and reduced in vacuo. The residue is triturated in water and the residue is triturated with ether. Mp. 173 to 176 ° C. and insolubles were filtered off. The FiI-Zit cleaning is dissolved in water and with Tiertrat is brought to pH 9 with soda solution and the carbon is treated. The clear water solution is extracted three times a precipitating oil with vinegar eggs. The concentrated liquid ester phases and the residue recombined from methanol-ether are crystallized with sodium sulfate 55. Yield 59.3g (63.6%). Mp. 197 to dried and concentrated. Fs remain 7.3 and a 199 C.
amorphous substance / mosquito (97.4 ° _n ). The substance is

chromatographically uniform. ^b » ^Z - ¹ ^ BOt) - Clv - NH - MhH

... ". _. _nn .,. _{m V1} "6.1c Z-Lvs- (BOC) ~ OH-DCHA (HmMoh

DZ- Phe - Phe - Pro - Pro he - Phe - \ al - Pro - _6o ^. "J ^ _{/ wipe vinegar creme,: o ml 2} " -citrene-

1 ro Ala -OtJu _{s ;; ure bQ} - _{() c verte} j | _t not Hssigcsterphase will tvit

. ν,, _r ^. ,,, ,, · ■■, χ ,,, ■ 2 ^ -citric acid and water, washed with Na-

aa) According to the Dicyclohexylcarbod.imid-X.-t ^ rHic nut _{iriumsillfilt} , _{ctdry and} eenct. The residue

Addition of 2 equivalents of 1 to 1 hydroxy-benzotriazole is _{dissolved in () ml of absolute} tetrahydrofuran. In addition

8.1 g of Z - Phe - Phe - Pro - Pro - OH CIIA (11 m-65 are added 1.5 g of 1-hydroxybenzotriazole (11 mmol) and added

Mol) between F.ssigester and 2 n-hydrochloric acid 0 ^: C a solution of 2.2 g DCC in cold DMF. Man

distributed. The ethyl acetate phase is left with water for 1 hour at OC and 1 hour at room temperature

to wipe. dried with sodium sulfate and concentrated. stand temperature and then gives 3.4 g

H-GIv-NH -MbH • HCl

(10 mmol) and 1.3 ml of N-ethylmorpholine (10 mmol) to. The mixture is stirred for 1 hour at room temperature and the precipitate is filtered off with suction. concentrates the filtrate. takes up the residue between warm ethyl acetate and sodium bicarbonate solution, shakes the ethyl acetate solution with 2η-citric acid, saturated sodium bicarbonate solution and water. dries with sodium sulfate and concentrates. The residue is recrystallized from ethyl acetate petroleum ether. Yield: 6.0 2 (90.7 ° ₀ ). Mp. 123 to 125 '

-8.9 ^: (c = 2, methanol).

O H - Lys- <BOCi - GIy - NH - Mbh · HCl

9.6 g of Z - Lys (BOC) -GIy-NH - Mbh are suspended in methanol, a palladium catalyst is added and 1N methanolic hydrochloric acid (auto-nitrator, pH 51) is added dropwise, and the catalyst is catalytically hydrogenated. The catalyst is filtered off with suction and the solution is concentrated. The residue is rubbed with ether. Bulge 7.3 e (S9%), recrystallized from methanol-ether. Mp. ~ 15S to 160 ^: C [\] d -17.55 (c - 2nd methanol).

d) Z - Pro - LyS (BOC) - GIy - NH - Mbh

To a suspension of 5 c Z - Pro - OH (20 mmol). 11.4 g of H - Lys- (BOC) - GIy - NH - Mbh ■ HCl (20 mmol) and 5.4 g of 1-hydroxybenzotriazole (40 mmol) in 60 ml of DMF are added to 2.6 ml (2OmMoI) N-ethylmorpholine and below Stir a cold solution of 4.4 g DCC in DMF. The mixture is stirred for 1 hour at 0 ° C. and 1 hour at room temperature, the NicderscNag is filtered off with suction. The feed is collected and processed as in 7. b). Yield 14.1 g (93 ° C.>. Mp. ISO to 183 C. Recrystallisicrable from methanol-water. [. \] D -27.4 ^: (c ---- 2. in DMF).

e) H - Pro - L \ s (BOC) - GIy - NH - Mbh · HCl

15.5 g Z - Pro · Lys. COC) - GIy - NH - Mbh were hydrogenated as in 7. c) in Methanoi kau ¹ ; .table, yield 15.2g (100 ° ₀ ). sinters from lÖO'C (amorphous).

f) NPS-AsN (MbJi) -OSu

18.6 g of NPS - AsN (MbJi) - OH · CHA (30.5 mmol) are given at 0 ^: C between ethyl acetate and about SOmI 2 η-citric acid. DL ethyl acetate phase is washed once more with 2η-citric acid and water, dried with sodium sulfate and concentrated. The residue is dissolved in 100 ml of tetrahydrofuran, 3.45 g of N-hydroxysuccinimide (30 m-mol) are added, the mixture is cooled to 0 ° C. and mixed with a solution of 62 g of DCC in cold tetrahydrofuran. The mixture is stirred for 1 hour at 0 C and 1 hour at room temperature, a little dimethylformamide is added. sucks off the precipitated dicyclohexylurea and concentrates the filtrate. The residue is boiled up with isopropanol. Yield 14.3 e (77 ⁰ Z ₀ "). Mp. 172 to 174 = C [λ] γ> ~ 15.3 ^: (r = -2nd DMF).

g) NPS - AsN (MbJi) - Cys (Mmb) - OH · CHA

To a solution of 2.25 g of H-Cyi (Mmb) -OH (8.2 mmol) in 20 ml of dioxane and 4.1 ml of 2N sodium hydroxide solution are added with stirring 2.5 g

NPS-AsN (MbJi) -OSu

and let stand overnight. The already neutral solution is concentrated and the residue is distributed between ethyl acetate and 2η-citric acid. Dropout, H - Cys (Mmb) - OH used in excess is filtered off. The ethyl acetate phase is once with 2 η-citric acid and washed once with water and dried with sodium sulfate. With cyclohexylamine the salt is felled. Yield: 2.45 sj (69.5%), m.p. 198-202X.

h) NPS - AsN (MbJi) - Cys (Mmb) - Pro - Lys (BOC) GIy -NH -Mbh

4.3 g NPS - AsN (Mbh) - Cys (Mmb) - OH · CHA

ίο (5 mmol) are ice-cooled between ethyl acetate and 2 η-citric acid distributed. The ethyl acetate phase is washed with 2 n-citric acid and water, dried with sodium sulfate and concentrated. The residue is dissolved in 20 ml of DMF. To the solution

give 3.15 g of H-Pro-LyS (BOCf-GIy-NH-Mbh ^ HCl (^ mmol). 0.64 ml of N-ÄtJulmcrpholin (SmMoI) and 0.7 g of 1-hydroxy-benzotriazole (5 mmol), cool to 0 ^C. and 1.1 g of DCC, dissolved in a little cold DMF, are added with stirring. The mixture is stirred for 1 hour at OC and 1 hour at room temperature, the precipitate is filtered off with suction and the filtrate is concentrated. The residue is taken up in ethyl acetate and shakes out as in 7, b). The residue is chromatographed on neutral aluminum oxide (Woelm, Aktiwufe I) in tetrahydrofuran. Yield: 4.95 £ (77 * _0). Mp. 160 to 164C [\] d-12.45 ^: ic = 2. D ^: methylformamide).

8. Synthesis of asparaginyl and glutaminyl peptides

a) Z - AsN - Leu - OCH _:)

To a solution of 2.7 g of Z - AsN - OH (IO mMoi 1.

1.7 g of H-LeU-OCH ₃ -HCl (10 mmol) and 2.7 g of hydroxy-benzotriazole in 20 ml of DNiF are added

OC 1.28 ml N-ethylmorpholine (10mMoH and finally a cold solution of 2.1 g DCC in a little DMF. The mixture is stirred for 1 hour at OC "and 1 hour at room temperature, the precipitate is filtered off with suction and the filtrate is mixed with water The precipitate is filtered off, triturated with sodium bicarbonate solution, filtered off with suction again, washed with water and dried over phosphorus pentoxide. Yield 3.35 g (5%), melting point 176 = C [\] v> -26.3 (c 2. methanol) .

b) Z-GlN -AIa-OBu '

To a solution of 2.8 gZ - GlN - OH (10 mmol),

1.8 g of H - AIa - OBu '■ HCI (10 mmol) and 1.35 g l-Hydroxy-1,2,3-b ~ n / iriazole (10 mmol) in DMF

one at 0 ^; C 1.2SmI N-ethylmorpholine (10 mmol) and finally a cold solution of 2.1 g DCC in a little DN1F. One learns now as in S. a). Yield 3.0 g (73.7%), m.p. 158 to 161 C [. \] D -36.0 ^: (c ■ - ■ 2, in methanol).

With the addition of 2 equivalents of 1-hydroxy-1,2,3-benzotriazole you got a yield of 2.9 g '71.2%). Mp. 158 to 16I = C.

9. Z-Ser-Gly-ONB To a solution of 2.4 g Z - Ser - OH (10 mmol), 2.7 c 1-hydroxybenzotriazole (2OmMoI) and 2.9 g H-GIy-ONB-HBr in 30 ml of DMF are added at ⁰ ° C. to 1.3 ml of N-ethylmorpholine and finally a cold solution of 2.2 g of DCC in DMF. One lets

Stir for 1 hour at room temperature and work as for TJ,, on. Yield 4.2g (97.5%). Melting point. 121 to 123 ° C h] D -8.2 ^D (c = 2, glacial acetic acid).

309 525/527

10. Corticotropin ^ l-23) -tricosapeptide-amide

S _r 25 g BOC - Ser - Tyr - Ser - Met - GIu (OBu ') - His Phe - Arg - Trp - GIy - OH

(5.5 mmol). prepared according to Chem. Ber .. 96 (1963), p. 1080. and 12.5 a (5 mmol)

H - LyslBOCi - Pro - VaI - GIy - Lys (BOC) - Lys (Boc) - Arg - Arg - Pro - VaI - Lys (Boc) - VaI - Tyr NH ₂ - tritosylate.

which was obtained from the acetate prepared according to Chem. Ber .. 97 (1964), p. 1197. by adding the calculated amount of toluenesulfonic acid in water, evaporating the solvent in vacuo and precipitating the residue from pyridine-ether, are in 150 ml DMF solved. After adding 2.7 g of 1-hydrolytic triazole (0 mmol), 6.5 g (30 mmoles) of DCC in 20 ml of DNiF are added to the solution at room temperature. After 1 hour a further third, after a further hour there :, the last third of the DCC solution is added. After 2 hours, the raw reaction product is dropped. with ether out. Yield 19.9g. The protecting groups are split off in a known ise W ^e by treating with lstündiges 90prozentiger Trifluoiessigsäure containing some thioglycolic acid, the crude Trikosapeptid WRD precipitated with ether and washed with Äiher. Yield 19.1g. For cleaning we. chrcmatoe uphiert in a known manner on carboxymethylcellulose.

11. Z - Leu - AIa - Leu - Glut UPu ') - GIv - Pro - Pro GIN -Ly S (BoO- Arc-OH of the solvent in the vacuum leaves a resin that is dissolved in ethyl ester and at OC with 2 n- Citric acid, bicarbonate and water are extracted. The solution is dried over Na ₂ SO ₄ and the solvent is distilled off in vacuo. The residue is a jelly which is triturated with ether and air-dried. Yield 106 g (89% TLC: pure. For analysis, a sample was crystallized from ethyl acetate ui. Melting point 130 to 132 ^D C, \ x \ i: -12.5 ^; (c = 1. in methanol).

, .H ₄₁ N-Oo (595.6)

"Calculate ... C 52.4, H 6.94, N 16.5 ^and "

found

C 52.4, H 7.1. N 16.7 '

and with

at T ₁ on

,! as: place.

a, - • C:

(Sequence 54-63 of porcine proinsulin) 11.5 g (ISmMoI)

Z - Leu - AIa - Leu - GIu (OBu ') OH

and 2.43 g (ISmMoI) 1-hydroxy-benzotriazole are mixed in 80 ecm DMF at -10 "C with stirring with 3.7 g (18 mmol) DCC in 25 ecm DMF. The mixture is stirred for a further 2 hours at room temperature, filtered from urea and gives the solution of 13.3 2 (15 mmol) H - GIy - Pro - Pro - GlN - Lys (Boc) - Era - OH _1. 1.5 CH ₃ COOH. H ₂ O and 1.92 ccm (15 mmol) N- Ethy morphine in 80 ecm DMF is added, the mixture is stirred for 2 hours at room temperature, the solvent is distilled off in vacuo and the residue is triturated with Essim, a crude yield of 17.1 g. The compound is boiled twice with ethyl acetate, the dried portion of sugar is thoroughly triturated with water three times . Yield 15.5g (73.5 ^O / _O ) - TLC: only traces of impurities are present. [Λ] ' ₀ °: -80.0 "(c = 2, in methanol) n

Amino acid ana! V.se

35

45

b) Z - Lys (Boc) - Arg (NO ₂ ) - OH (581.61

89 e (0.15 mol) of the one prepared according to a). F.5 are dissolved in a mixture of 240 ecm Was.-er 500 ecm Diexan. The ISO ecm 1 N NaOH solution is added, the mixture is stirred for 45 minutes at room temperature and made neutral with 30 ecm 1 N HCl. Dioxane is distilled off in vacuo, the VV phase is extracted twice with ethyl acetate and acidified at OC with 150 μm In-HCl. Γ · an oil separates. the soak in ethyl acetate. will. After washing the solution with water, the solvent is removed with the addition of benzene. The warm residue solidifies on trituration with petroleum ether. Yield 79g (91%). TLC: Fa.-t: trace ester. Sample boiled with ethyl acetate: pure, m.p. 16S to 171 - "C N '?: -4.8' (c methanol).

Co-, Η ,,, Ν-Οο (581.61

"Calculated ... C 51.6. H 6.76. N 16.86%: Found ... C 51.6, H 6.8, I. '6.7%.

c) H - Lvs (Boc) - Ars - OH, 2 CH ₃ COOH. H ₂ O (540)

75.5 c (0.13 mol) of the dipipe obtained according to b) are dissolved in 800 ecm acetic acid-methanol-Va ^ r (4: 4: 2) and catalytically hydrogenated on Pd. After the catalyst had been filtered off, the filtrate was evaporated in vacuo, the residue was triturated with ether and _{evaporated over P 2} O ₅ , the residue was triturated with ether _{and dried over P 2} O ₅ and KOH. The acetic acid content depends on the drying conditions. Yield 70 g (almost quantitative).

Acetic acid

Calculated for 2 moles ... 22.2 '

/ 0.

Calculated
found

Calculated
a found

GIu ₂ Pro ₂ GIy ₁ AIa ₁ Leu ₂ Lys, Arg, GIu _2i05 PrO ₁ , _M Cly _o , ₉₈ Ala _liO j Leu _{2 co} LyS _1-03 Arg _09:.

1 equivalent
1.09 equivalents.

found 21.8%.

water

Calculated for 1 mole ... 3.34%;

found 3.7%.

d) Z-GIN-LyS (BOC) - Arg - OH (664)

Production of the starting products a) Z - I vs (Boc) - Ar ₈ (NO ₂ ) - OCH ₃ (596)

57 g (0.2 mol) H - Arg (NO ₂ ) - OCH ₃ · HCl are stirred with 95 g (0.2 mol) Z - Lys - (Boc) - OSu in 500 com DMF overnight. After 70 g (0.13 mol) of the dipeptide obtained in c) have been distilled off, 60 g (0.15 mol) of Z-GlN are added to 600 ecm of DMF with the addition of 16.5 ecm (0.13 mol) of N-ethylmorpholine -ONp stirred at room temperature for 24 hours. The solvent is distilled off in vacuo, the residue is boiled with ethyl acetate and the insoluble peptide is filtered off while hot. For further purification, it is dissolved in 150 ecm of methanol and the

Solution with 750 ecm 1 of n-NH ₃ , whereupon the tripeptide crystallizes out. Yield 66.5 g (82 ° / “). DC: Pure.

e) Z-PrO-PrO-GlN-LyS (BoC) -Arg-OH (858)

The Z-tripeptide (0.1 mol) prepared according to d) is catalytically hydrogenated in 350 ecm DMF with the addition of a large amount of Pd (BaSO _1). The splitting off of the Z group has ended after 2 hours. 62 g (0.115 mol) of Z - Pro - Pro - OSu in 80 ecm DMF are then added and the mixture is stirred for 12 hours at room temperature. After the solvent has been distilled off in vacuo, the residue is boiled with ethyl acetate, triturated with fresh ethyl acetate and washed with ether. Yield 79.9g. TLC: The pentapeptide still contains about 10 ⁰ Z ₀ Pyr-Lys (Boc) - Arg - OH. the end

Yield after filtering off the catalyst, evaporating the filtrate in vacuo, triturating the residue with ether and drying over P ₂ O ₅ and KOH: 35 g (97 ° / ₀ ). The acetic acid content is not stoichiometric and varies with the drying conditions.

i) Z - AIa - Leu - GIu (OBu ') - OCH ₃

H - GlN - Lys (Boc) - Arg - OH

arose and cannot be separated at this stage. However, it does not occur in the following conversions Pe.iction and can easily be done at a later stage be separated.

Z - Pro - Pro - OSu is represented as follows: 69.2 g (0.2 mol) of Z - Pro - Pro - OH are suspended _{in 500 ecm CH 2} Cl _2. 27.6 g; 0.24 Mc!) HOSu are added. stirs for 10 minutes, cools to −5 ° C. and adds 41.2 g (0.2 mol) of DCC in 100 ccm of CH ₂ Cl ₂ . After stirring for 30 minutes at 0 ° C. and 2 hours at room temperature, the urea is filtered off and the mixture is brought to 0 ^; C cooled solution of 26.5 g of Z-Leu-OH (0.1 Molf and 27 g of 1-hydroxy-benzotriazole (0.2 Mo!) In 200 ml of tetrahydrofuran are added 25.4 g of H - GIu (OBu ') - OCH ₃ · HCl, 12.8 ml of N-ethylmorpholine (0.1 mol) and finally a cold solution of 22 g of DCC in a little tetrahydrofuran The mixture is stirred for 1 hour at OC and 1 hour at room temperature, the precipitate is filtered off with suction and worked the! urate as in II. \ . The residue is dissolved in ethyl acetate over basic aluminum oxide (W oe I ni.

Active stage 1) Chromatographies The eluate is concentrated in vacuo. Yield: 44.85 g of oil (96.6 " ₀ ). The above oil is catalytically hydrogenated in methanol as in VI. S. The resulting Dipeptide sterhydrochvid is oily. Yield: 34.8 g (95 mmol). This oil is mixed with 21.2 g of Z - Ala-OH (95 mmol) and 26.6 g of 1-hydroxybenzotriazole (19Ommol) dissolved in 200 ml of DMF. At OC, 2.2 ml of N-ammonium morpholine (95 mmol) and a cold solution of 20.9 g of DCC in DMF are added I hour at OC and 1 hour. · At

the filtrate to dryness in vacuo. The resinous stand at room temperature, the precipitate is sucked off. The residue is dissolved in 500 ecm of ethyl acetate, quickly and the filtrate is worked up as in II. 1. The back at 0 "C is washed with bicarbonate and water and is _{dried in tetrahydrofuran over a basic over Na 2} SOj. The solution is chromatographed in vacuo with aluminum oxide. The eluate

brought to dryness and solvent in a high vacuum! freed. Yield 71g (80%).

f) H - Pro - Pro - GlN - Lys (Boc) - Arg - OH

71 g (S2.6 mmol) of the Z compound are catalytically hydrogenated on Pd in 800 ecm of methanol. After the solvent has been distilled off in vacuo, the residue is triturated with ether. Yield 58.2 g (97.5 ^c "/ ₀ ). TLC: Contains only the Pyr - Lys (Boc) - Arg - OH formed under e) as an impurity.

vom is concentrated and rubbed with petroleum ether. Yield 40 2 "(74.7 ° _0. Bezocen on Z -Leu -OH) m.p. 96 * to 9SC, [\] d -" 54.1 (c = 2nd methanol), 724'-

k) Z - AIa - Leu - GIu (OBu ') - OH

15.85 g of Z- AIa - Leu - GiuiOBu ') - OCH ₃ (28.65 mmol) are added to a mixture of 100 ml of dioxane and 16 ml of water. With stirring, 29 ml of 1N sodium hydroxide solution are slowly added dropwise over the course of 2 to 3 hours (thymolphlhalein as indicator). It is then neutralized with 2η-citric acid and concentrated. The residue is divided between 2 normal itric acid and ethyl acetate. The ethyl acetate solution is washed once with water, with sodium sulfate

DMF with 34.8 g (90 mmol) Z - GIy - OTCP dried for 24 hours and concentrated. The residue will be off stirred at room temperature. The liquid ester is distilled — petroleum ether crystallizes. Yield: 14.05 g

The solvent is removed in vacuo, the residue is dissolved in 100 ecm of methanol and the reaction product is precipitated with 1 1 ethyl acetate. For cleaning, it is extracted several times with warm water. The aqueous extract is filtered and evaporated in vacuo, the back

c) Z - GIy-Pro-Pro-GIN-Lys (BoC) -Ar -OH (916)

36.2 g (50mMoI) pentapeptide are in 200 ecm

(91 ⁿ / ₀ ), m.p. 142 to 145 ^: C. Recrystallized from ethyl acetate peuol ether: m.p. 145 to 14SC [\] d - 45 ^; (c = 2, methanol).

stood again from methanol-ethyl acetate reprecipitated. Yield 37, L g (81%). DC: Except

Pyr - Lys (Boc) - Arg-OH no further impurity.

h) H - GIy - Pro - Pro - GlN - Lys (Boc) - Arg - OH, 1 ■ 5 CH ₃ COOH, H ₂ O (890)

The Z compound obtained according to f) is catalytically hydrogenated in 300 ecm of 90 percent acetic acid on Pd.

1) Z - ^T .eu - AIa - Leu - GIu (OBu ') - OH

11g Z-A! A-Leu-Glu (OBu ') - OH are in a mixture of 50 ml of methanol and 50 ml of glacial acetic acid and dissolved after the addition of palladium catalyst catalytically hydrogenated. After the hydrogenation has ended, the catalyst is filtered off with suction and the filtrate is concentrated and the residue triturated with ether. Yield 7.95 g, m.p. 214-215 "C.

The hydrogenation product is suspended in 50 ml of DMF. 7.25 g of Z - Leu - OSu (20 ° / ₀ excess) are added and the mixture is stirred for 1 day at room temperature. The solution is concentrated and the residue

65

between ethyl acetate and 2 η-citric acid distributed, the ethyl acetate phase is washed with water, with Dried sodium sulfate and concentrated. From ethyl acetate — petroleum ether is recrystallized. Yield 10.45 g (82%). Mp. 186 to 189 ° C [*] d -49.5 ° (c = 2, in methanol).

12. Other isolated 1-hydroxy-benzotriazol esters a) Z-GIy-OBT

As in Example 5, a), 2, IgZ-GIy-OH (10 mmol), 1.5 g of 1-hydroxy-benziiazole (11 mmol) are reacted in tetrahydrofuran with 2.2 g of DCC. Yield 1.6 g, mp. 147 ⁰ C (from isopropanol).

b) Z - Thr - OBT

As in Example 5, a), 2.6 g of Z-Thr-OH (10 mmol), 1.5 g of 1-hydroxy-benzotriazole (11 mmol) are reacted with 2.2 g of DCC in tetrahydrofuran. Yield 2.5 g, mp. 150 to 153 ⁰ C (from isopropanol).

c) Z-AsN (Mbh) -OBT
As in Example 5, a) 4.6 g of Z-AsN (Mbh) -OH

(10 mmol), 1.5 g of 1-hydroxy-benzotriazole (UmMoI) reacted with 2.2 g of DCC in DMF. Yield 4.0 g, mp. (Boiled with isopropanol) 158 ⁰ C.

1 sheet of drawings

Claims (1)

Claim: Process for the preparation of peptides by reaction of a protected amino acid or a protected peptide in which the carboxyl group that is to react is released is, with a protected amino acid or a peptide ester or amides thereof, in which the Amino group that is supposed to react is free, in a common in peptide chemistry Solvent in the presence of a carbodiimide or by reacting a protected one Amino acid or a protected peptide in which the carboxyl group involved in the reaction should occur, is free, in the presence of a carbodiimide to an activated derivative and then Implementation with a possibly protected pep! ·.! or one, amino acid or egg amides, in which the amino group that is to react is free. in one in peptide chemistry Usual solvents, where appropriate after the end of the reaction and the usual cleaning all or some of the protective groups be aged in a known manner, characterized in that one the reaction is carried out with an addition of 1 to 2 equivalents of 1-hydroxyl-benzotriazole. One of the most convenient methods of 2.α production of peptides consists in linking an N-acylamino acid or an N-acylamino acid or peptide ester with an amino acid or peptide ester by means of diclohexylcarbodiimide (JC Sheehan and GP Hess, J. Amer. Chem. Soc . 77 [1955], p. 1067). The disadvantage of this method is the not inconsiderable racemization when linking peptides (F. Weygand, A. Prox and W. K ö η ic, Chem. Ber., 99 '[1966], pp. 1451 to 1460) and the Formation of N-acylureas, which contaminate the synthesis product and reduce the yield (see E. Schröder and K.Lübke, The Peptides, Vol. I, pp. 108 to 111, New York and London 1965). Additives \ on 1.1 to 2 equivalents of N-hydroxysuccinimide for peptide synthesis with dicyclohexylcarbodiimide reduce racemization to less than 1 ° / ₀ D compound and prevent the formation of N-Acy'harnst.offen (F. W EYCA η d, D. H ο ffma η η, E. Wü η sch, Z. Naturf., 21 b [1966], pp. 426 to 428; JE. Zimmermann and GW A η ders ο η, J. Amer. Chem. Soc, 89 [1967], p. 7151). However, the N-hydroxysuccinimide additives in the dic \ clohe. \ Y! Carbodiimide method also have serious disadvantages. M. L ö w and K isfa 1 udy (Acta. Chim. Hung., 44 [1965], pp. 63 to 65) reported that N-hydroxysuccinimide itself reacts with picyclocarbodiimide and that sterically hindered N-acyl peptide-N-hydroxysuccinimide -est were not producible. A compound of 1 mole of dicyclohexylcarbodiimide and 3 moles of N-hydroxysuccinimide was later used as succinimidooxycarbonyl - ^ - alanine hydroxysuccinidesier erv, N. (H. Gross and L. Bilkin E. Bricas: Peptides, North-Holland Publishine Comp. [1968], p. 156 and 157). This compound reacts smoothly with amines to form urea derivatives of the ^ -alaninamides. Has been confirmed this finding by F. W e y g a η d, W. S t e g 1 i c h and N. C h y t i 1 (Z. Naturforsch., 23 b [196S], P. 1391). In the implementation of BOC-L-glutannic acid -_ \ - benzyl ester with N-hydroxysuccinimide and ίο dicyclohexylcarbodiimide and subsequent addition of 2.4,6-trimethoxybenzylamine was a considerable quantity of succinimido - 2,4,6 trim.ethüxybenzylamid isolated - oxycarbonyl -beta-alanine. It has now been found, surprisingly, that with the dicyclohexylcarbodiimide method, an addition of 1-hydroxy-benzotriazole (HOBT) also reduces the racemization in the racemization test by Weygand and coworkers (Chem. Ber. 99 [1966], pp. 1451 to 1460) under l ° ' _o d-connection lowers and the N-acylurea formation \ reduced (see table 1 [example \, I]). In addition, no by-products can arise, as are possible with the addition of N-hydroxysuccinimide. The yields are therefore generally significant with the addition of I-hydroxybenzotriazole higher, and the isolated compounds are obtained in a purer form. The invention thus provides a method for producing peptides by reacting a protected amino acid or a protected pep- lids in which the carboxyl group that is to react is free, with a protected amino acid or a peptide ester or their amides in which the amino group. which react should, is free, in a:, n commonly used in peptide chemistry Solvent in the presence of a carbide diimide by reacting a protected amino acid or a protected peptide to which the carboxyl group that is to react is is free, in the presence of a carbodiimide to an activated derivative and subsequent reaction with an optionally protected amino acid or an optionally protected peptide b / w. whose Amides, in which the amino group. that is supposed to react is free, in one of peptide chemistry common solvents! after completion of the reaction and customary cleaning, if necessary the protective groups are completely or partially split off in a manner known per se, which is characterized by this is that the reaction with an addition of 1 to 2 equivalents of 1-hydroxy-benzotriazole performs. N-protected amino acids and peptides can be used with 1-hydroxybenzotriazole and dicyclohexylcarbodiimide produce activated products that are eminently suitable for peptide synthesis. Eliminate it here »activated esters«, which in solution are obviously in equilibrium with an »activated amide« stand. Depending on the substance, the “activated ester” or the “activated amide” can be isolated. Ir Usually, however, it is not necessary to isolate the activated product. —CO - OH Dicyclohexylcafbüdiimide R - - CO - O - N OH