DE2209835C3 - Insulin derivatives, processes for their production and medicaments containing them - Google Patents

Description

is linked, where X stands for a direct carbon-carbon bond, an aliphatic hydrocarbon chain with 1 to 15 carbon atoms, in which one or more carbon atoms are optionally replaced by heteroatoms or heteroatoms, and their further valences either only with hydrogen atoms or with hydrogen - utomes and hydrophilic groups are saturated.

2. Process for the production of bifunctionally crosslinked insulin derivatives in which the · amino group of glycine * ^{1 of} the Α-chain with 4ct f-amino group of lysine ^{029 of} the B-chain through a dicarboxylic acid bridge of the formula

—C — x — c—

(I)

Is linked, where X door is a direct carbon-carbon bond, an aliphatic hydrocarbon chain with 1 to 15 carbon atoms, in which optionally one or more carbon »Tome through heteroatoms or heteroatom groups (pen are replaced, and their further valences either only with hydrogen atoms or with hydrogen atoms and hydrophilic groups are saturated, characterized in that Man insulin with activated derivatives of dicarboxylic acids of the general formula

Y-C-X-C-Y

III)

Where X has the meaning given above, And Y for a carboxylic acid group activating The remainder is in polar organic solvents or in a mixture of organic solvents Reacts with water, in the presence of proton acceptors under dilution conditions, and the reaction products obtained isolated by separation processes, on the one hand according to the molecular size and on the other hand according to the charge differentiate.

3. Medicament, characterized by a content of at least one insulin derivative according to Claim 1.

60

It has already become known that insulin is chemically modified by means of monofunctional reagents It is possible that derivatives represented in this way can also be obtained in a uniform form d that such insulin derivatives have changed biological properties (see for example!

835

D. Levy and F. H. Carpe η t er. Biochemistry, 6. 3559 [1967]; D. G. Lindsay and S. Shall. Biochem. J., 121, 737 [1971]; B.Africa and F. H. Carpenter, Biochemistry, 9, 1962 [1970]).

about a therapeutic use of these reaction products however, nothing has yet become known of insulin with monofunctional reagents.

When converting insulin with bifunctional reagents, only heterogeneous mixtures have so far been possible and no uniform reaction products are obtained (see, for example, DL-PS 10002; H. Zahn and J. Meienhofer, Makromol. Chem., 26, 153 [1958]). The reagents always reacted with different functional groups of the insulin molecule, and, in addition to monomeric derivatives, there were also higher molecular weight products with an indeterminate amount Degree of polymerization (see for example DL-PS 10002).

The two previously known uniform insulin derivatives with an intramolecular m-phenylenedithiocarbamoyl bridge proved to be biologically inactive (D.Brandenburg, H. G. Gattner. M. Weinert, L. Herbertz. H. Zahn and A. Wo linier. Diabetes. Proc. 7th Congress Im. Diabetes Fed. Buenos Aires. 1970. Excerpta Medica Int. Congr. Series, 231, 363 [1971]}.

It has been found that the new, bifunctionally crosslinked insulin derivatives, in which the <i-amino group of glycine ^{A1 of} the Α-chain with the> -amino group of lysine ^{829 of} the B-chain of the insulin molecule through a dicarboxylic acid bridge of the formula

C-X-C—

(I)

is linked. where X is a direct carbon-carbon bond, an aliphatic hydrocarbon chain with 1 to 15 carbon atoms, in which optionally one or more carbon atoms have been replaced by heteroatoms or heteroatom groups, and their further valences either only with hydrogen atoms or with hydrogen atoms and hydrophilic groups are saturated, have a high sugar-lowering effect.

It has also been found that crosslinked insulins in which the u-amino group of glycine ^{A1 is} linked to the f-amino group of lysine by a dicarboxylic acid bridge of formula 1 are used. where X has the meaning given above, is obtained when insulin is used with activated derivatives of dicarboxylic acids of the general formula

Y C - X - C - Y

(II)

where X has the meaning given above and Y is one which activates the carboxylic acid group The remainder is in polar organic solvents or as a mixture of organic solvents Water, in the presence of proton acceptors under dilution conditions and the resulting Reaction products isolated by separation processes, which once according to the molecular size and for differentiate others according to the charge.

Surprisingly, the invention

According to the crosslinking reaction, only intramolecularly crosslinked insulin derivatives are formed in which the amino groups of glycine * ¹ and lysine " ^{29 are} linked. Thus, the temporary protection of the phenylalanine ^B1 amino group could be dispensed with, although it had to be expected according to the prior art that this would Amino group would be substituted to a large extent (DG Lindsay and S. Shall Biochem. J-, 121, 737 [1971]; D. Levy and FH Carpenter, Biochemistry, 6. 3559 [1967]). It is also unexpected that side reactions on tyrosine - and serine OH groups (H. Zahn and F. Schade, Angew. Chem., 75, 377 [1963]) did not occur.

It is also very surprising that the inventive Insulin derivatives have a higher blood sugar lowering effect than most monofunctional cells

S S

H ₂ N-GK Cys -

C \ s have substituted, known insulin derivatives and a significantly higher blood sugar lowering effect than the previously known bifunctional cross-linked have insulins.

The unexpected finding that the insulin derivatives according to the invention are made from zinc salt-containing Solutions can be crystallized. This gives the possibility of an application in form of crystal suspensions, which are known to be applied to insulin with a view to a protracted effect will.

The substances according to the invention thus represent an enrichment for pharmacy.

Use bovine insulin and bis-p-nitrophenyl adipate as starting materials, the reaction sequence can be represented by the following simplified equation:

C \ s - Mn

A chain

H ₁ N- Phe

+ CXN-

SS Nh,
C \ s Cys Lys-AIa

19th 3 (1

/ - O — C- (CH, I ₄ -CO - /
OO

B chain

2 0, N-

-OH

S-

HN- ^ GIy Cys-Cys

C \ s

Cvs-Asn

H, N-

Phe

Cvs

OS
Cvs

NH
-Lvs - AIa

.1 1

In formulas I and II, X is preferably - (CH ₂ ) "-, where η can be an integer and zero. Y preferably represents optionally substituted phenoxy.

The bifunctional compounds which can be used according to the invention are already known or can be prepared by known processes (H. Zahn and F. Schade, Chem. Ben. 96, 1747 [1963]; J. Schnell and H. Zahn. Colloid-Z .. 203, 27 [1965]; H. Zahn and M. Bahra. Research area of the state of North Rhine-Westphalia. No. 1897; L.Brandt. Ed., West German Verlag, Cologne and Opladcn [1967]). usually from the dicarboxylic acid, the substituted phenol and Dicyclohexylcarbodiimide.

Examples are:

Derivatives of aliphatic dicarboxylic acids of the general formula

YOC- (CH ₂ I _n -COY
in which the carboxyl group is activated by the radical Y, such as adipic acid-bis-p-nitrophenyl ester. Pimelic acid bis-N-hydroxysuccinimide ester, suberic acid - bis - 2,4,5 - trichlorophenyl ester, sebacic acid bis-pentachlorophenyl ester. as well as amino acids which have two carboxyl groups in activated form and in which the amino group (s) is protected, such as N.N'-bis-tert.-butyloxycarbonyl-cystine-bis ^ AS-trichlorophenyl ester, N - benzyloxycarbonyl - glutamic acid-ci, ; -bis-p-nitrophenyl ester.

As insulins, for example, can be used; Beef insulin, pig insulin. Sheep insulin. W ^ .insulin, fish insulins.

All polar organic diluents can be used as diluents Solvents in which insulin dissolves, as well as mixtures of these solvents, can be used with water. Examples are dimethyl sulfoxide and dimethylformamide.

hs All can be proton acceptors in peptide synthesis common basic compounds can be used, e.g. B. organic bases such as triethylamine or N-methylmorpholine (the latter preferably

when working with optically active carboxylic acid / ur Avoidance of racemization), also alkali hydroxides when working in the presence of water. Hydrogen carbonates or carbonates such as sodium hydrogen carbonate or sodium carbonate.

In general, temperatures between 0 and 40 ° C., preferably 18 to 25 ° C., are used.

The reactions are usually carried out at normal pressure.

When carrying out the process according to the invention, 1 mol of crystalline insulin is used amorphous insulin 1 to 2 mol, preferably 1.2 to 1.3 mol, of the activated dicarboxylic acid derivative. in such a way that the solution of the activated derivative slowly dripped into the dilute solution of the insulin is, usually for 1 to 5 hours. The reaction mixture is then left for several more Hours, optionally with stirring. Light and light are cheap, but not necessary Oxygen exclusion, d. H. Working in the dark and under protective gas (e.g. nitrogen) to avoid this side reactions (e.g. oxidative damage to insulin).

The work-up is carried out either by precipitating the insulin derivatives by adding with the Solvent-miscible solvents which have the property of precipitating the insulin derivatives, for example methanol and ether. You can also counter the reaction solution in the dialysis tube Dialyze water to separate the insulin derivatives. The reaction can be terminated before the start acidified during dialysis, e.g. B. with acetic acid. Dialysis against dilute patients has also proven to be beneficial Ammonium bicarbonate solution has been shown to leave residual activated groups of the dicarboxylic acid aminolyzed.

After dialysis, either direct lyophilization takes place. or the insulin derivative is made by cessation of the pH value precipitated to the isocleklrischcn point.

The preparation is then either moist or, after drying, fractionated by means of a process, separates the molecules according to their molecular weight, preferably by means of Gclchromatographie Conditions under which insulin and the derivatives do not aggrcate. For example, here is Sephadex G-50 line used in 10% acetic acid. The preparation obtained after dialysis and freeze-drying (Monomer fraction 3. see examples) is further fractionated according to the method, the molecules according to the

<■ Disconnect the charge. This can be done by ion exchange malography or electrophoretic processes are used. Preferably in the acidic medium is a ion exchange chromatography applied, e.g. B. on SE-Sephadex A25 at pH 3.0 in acetic acid 7 M urea with an NaCl gradient.

That after dialysis. Freeze-drying and (possibly necessary) desalination by means of gel filtration. /. B. to Scphadcx G 25. or product obtained by isoclectric precipitation, if necessary.

is by ion exchange chromatography in weakly alkaline solution. /. B. on DEAE-Sephadex A 25 at pH 8.4. be cleaned afterwards.
The following new active ingredients are mentioned in detail:

N ' ^M .N' ^B2u -oxalylinsulin (bovine).
N ' ^M .N "^{);! 9} -Succinylinsulin (bovine).

_Ν ιλι.N '^ - Glutarylinsulin (beef).

N ' ^M .N ^rH2IJ -Adipoylinsulin (Rind. Schal ").

N ' ^M .N ^IB2g -pimcloyl insulin (bovine).

N ^3M .N ^{11) 21J} -uberoyl insulin (bovine).
^ N ^lA1 .N ^{Eli: 9} -Azelaoylinsulin (beef),

N ' ^M .N ^rB29 -ScbacoyIinsulin (Bovine. Pig).

N ' ^A1 .N'" ^2U -undccandioyl insulin (bovine).

N ^lM .N ^rB2q -dodecanedioyl insulin (beef)

N ¹ * ¹ .N ^fB2q -Tridecanedioyl insulin (bovine).
ίο N ' ^M .N ^r " ²⁹ - (NN-bis-tert.-butyloxycarbonyl) -

i -cystinyl insulin (bovine).

_n , ai.N ^rH29 -i-cystinyl insulin (bovine).

N ' ^M .N ^fli2 "-N-benzyloxycarbonyl-L-glutamyl-

insulin (beef).
i> N ^lM .N ^rB - ^g -i.-Glulamylinsulin (bovine).

Chemical structure evidence for the covalent
networked insulin derivative c

a) Insulin and all derivatives in which amino groups Are monofunctionally substituted, give in the cleavage of the disulfide bonds. z. B. by oxydalive Sulphitolysc (L. B a i 1 c y and R. D. C ο 1 e J. Biol. Chemistry. 234, 1733. [1959]). the separated A and B chains as S-sulfonates. Simplified: Scheme:

Sulphitohse
AB (SS)., - - AlSSO ₁ U -i B (SSO ₁ I;

B-chain c

insulin

A chain c

In the case of an insulin derivative linked by means of a bifunctional bridge between glycine "and phenylalanine" ¹ or lysine " ²¹ 'ν only one chain derivative may occur after the cleavage:

-AB (SS)
V O Sulphitohse -A (SSO, U
OC ν Λ
ο I.
X insulin OC-B (SSO, I, \ ernct / tcs \ ernctztcs chain derivative

All preparations (examples J to 4, 6 to 9) were s ₅ gave two bands, as expected, since c

subjected to a Sulphitolysc, the electrophoresis newly introduced bridge is cleaved,

showed only one band (see Fig. 1. No. 2). Only those with b | Proof that the bridge is exclusive

Cystine crosslinked derivatives (Examples 11 and 12) borrowed between glycine ^M and lysine ^B2l) and not zv

see glycine ^Λ1 and phenylalanine '", succeeded by enzymatic degradation of the crosslinked Celtic derivatives by means of trypsin and is confirmed by end group determination.

Adipoyl-A-ketlen-telra-S-sulfonate-B-chain-bis-S-sulfonate was with trypsin according to S. S. W a η μ and I · '. 11. Carpenter. Biochemistry. 6. 215 (1967). split. The cleavage products were after freeze-drying by paper electrophoresis pH 2 (2.4 M formic acid 7 M urea! separated (see Fig. 1. No. 3).

Only the two cleavage peptides given in the following table were found. All other cross-linked insulins (Examples 1 to 4 and 6 to 9 | gave in the electrophoretic analysis and Lud group determination same results.

The following table shows the amino acid composition of the derivative Ν ^7λ1 .Ν " ^ll obtained from adipoylinsulin by oxvdalivc sulphitolysis: '' -Adipoyl -A -chain-tetra-S-sulfonate-B-chains -bis-S-sulfonate (A - Ad B) and the resulting iryptic cleavage peplides Ν ^ϊΛΙ .Ν ''"" - adipoyl A-chains - tetra - S - sulfonal - B (23 to 30) [designation: A Ad B (23 to 30)] and B ( I to 22) -bis-S-sulfonate [designation: B (I to 22)].

Amino acid analysis according to Moor e and Stein. Duration of hydrolysis: 48 hours at 110 ° C. All values are uncorrected and refer to glycine.

amino acid A \ il Ii A Vl Ii ii (23 to 3 (left (I hi- LVS COME ON 0.97 0 HIS 2.05 0 ARG 1.04 0 1.16 ASP 3.43 1.95 0.81 HER 1.13 1.07 1.69 SI-R 2.S0 1.73 I.2S GFU 6.S0 3.72 1.34 PER 1.48 j 0 GLV 4.00 2.00 2.00 ALA 3.28 2.00 0.90 Semi-cystine 4.72 3.61 ■ i VAL 5.06 1.70 4.05 ILI 0.70 0 0 LHU 6.33 1.91 3.70 TYR 4.10 2.68 0.71 PHL 3.14 2.03 1.50 The / calibrate J- means present, letloch flour quar hcsiimml.

40

50

55 s

B (2.1 to 30) - amino acid sequence 2.1 to 30 of the H-Kclle
Ullhis22l = amino acid sequence 1 to 22 of the B-KeIIe

Description of the FIG. 1 to 4 of the patent application

The fi and 1 have four paper telephones at pH 2 "(buffer: 2.4 M HCOOH 7 M urea) of:

1. A-Kcttc iiinksi and B-K et ic (right) in the S-sulfonate form.

2. N ' ^M .N " ^12IJ -Adipoyl-A-chain-tetia-S-.nulfonate-B-chain-bis-S-sulfonai.

3. the gap obtained therefrom by decomposition of I rypsm pepliden and

4. the one made from insulin-B-Celts-S-sulfonate with Tnpsir preserved Spallpepli Jen. B II to 22) (left) around B (23 to 29) (right).

This was stained with dia / otierter sulfanilic acid. The arrow mark should be the starting line de I mark lekirophoresen.

Figure 2 shows the ulion diagram of the gel Chromatography of about 600 mg of crude Azelaovl insulin (Example S) on a column (dimensions 5 χ 150 cm) with Sephadex G-50 line in 10 "uigei Acid flow rate 100 ml hour Fractions of 12 ml.

Abs / isse: parliamentary group number.

Ordinate: F. absorbance at 254 µm.

Fraction 1 contains oligomcrc. Faction 2 at all dine neutrally. Fraction 3 monomeric insulites father. The flow diagrams of the gel chromatography pll ic of the other hi-functionally cross-linked insulin derivatives show a similar trend.

F i g. 3 / shows the flow diagram of the ion exchange chromatography of 320 mg of raw silver ovlinsulin (Example 7. Fraction 3 | on a column (Dimensions 1.5 45cm) with SL-Sephadex be pH 3.0 Linear sodium chloride gradient. Buffer sees Details in Example I. Duration 30 to 35 ml hour. Fractions of 6.8 ml. I.xlink tion measurement at 254 nm.

Paragraph: section number.

Ordinate: NaCI con / entration in minor.

Fraction with the maximum at fraction number 60: the insulin derivative sought with a bridge between glycine ^M and lysine " ^24. The evolution diagrams of the ion exchange chromatography of the other bi-functionally crosslinked insulin derivatives show a similar course.

F i g. 4 shows the UV spectrum of amorphous

Insulin (-). Concentration = 0.767 mg ml

and glutarylin insulin (). Concentration

= 0.636 me ml. In 0.05 M ammonium bicarbonate solution. pH p.2.

Ordinate: Hxtinklion.

Abscissa. Wavelength in nm in nanometer.

The new active ingredients show a blood gliicosis lowering effect on. They are therefore suitable for: Treatment of diabetics in general uiu especially those caused by antibodyhikiuni need high doses of conventional insulin preparations against beef and / or porcine insulin However, experience has shown that after switching to a new preparation, against which there are no symptoms are formed, is covered by lower doses satisfactorily.

The new active ingredients can be converted into the customary formulations in a known manner such as solutions and suspensions, especially crystal suspensions.

The new active ingredients can be used in the usual way, in particular for parenteral purposes Injection.

Versuchsanordnuivj to examine the insulin it was from! nsi'.üvsderivaien

Insulin lowers the blood glucose \ on rat dicm Effect is dose-dependent in a certain range hillside!;.' By comparative Heredininvj dei BI111

22nd

Glucose lowering in rats after Suhcuian injection of preparations with unknown insulm efficacy and blood glucose lowering after subeutaneous injection of insulin with a known active ingredient content is the blood glucose-effective insulin activity of the insulin derivatives determined. For details see H. Zahn. E. D r e c h s e I and W. Puls: I loppe Seylers Z .. Plnsiol. ("hem. 349. 3X5 to 3X9 (1968).

In the next section, the blood glucose will be effective Insulin activity of some compounds according to the invention as a percentage of the insulin standard 125.4 Il mg) stated:

ΒΙιιίμΙιιι'ΟΜ * - vveffectivec lnsuünakliviläl 1 mg in percent of insulin landard (25.4 IU mgi

_N , v. _N , »». Oxaiyijnsuijn, Rj _nc i)
N'M.N'-i '^. Giutarylinsulin (Rindl
N ^3V '.N ^rluu -Adipoylinsulin (beef)
N ^3A '.N ^rH29 -Pirneloylinsulin (bovine)
N ' ^M .N ^clul) -Suberoylinsulin (beef)
N ^3M .N ^fB ;! Q -azclaoyl insulin (beef)
N ' ^{and N r B2q} -Sebacoylinsuiin (beef)

52

32

41

35

100

100

61

example 1
N ^IAl .N ^cBM -oxalylinsulin (bovine) JS

A solution of 640 mg (ΙΟΟμΜοΙ) crystalline Beef insulin and 150μ1 triethylamine in 75 ml dimethyl sulfoxide was added dropwise with a solution of 39.8 mn while stirring at room temperature (120μΜο1) bis-p-nitrophenyl oxalate in 5 ml Dimethyl sulfoxide was added within 4 hours. The reaction solution stood still for 60 hours Room temperature and was then initially for 2 hours against running water and then two- ■ lal each dialyzed for 1 hour against 1 1 0.05 M ammonium toicarbonate solution. The solution was with dilute HCl acidified to pH 4.9, the precipitated protein is centrifuged off and washed with water. The moist product was dissolved in 3 ml of ice cream, vinegar and 17 ml of water and placed on a column <5 χ 150 cm) with Sephadex G-50 fine in 10% acetic acid chromatography. The eluate was in 3 fractions (see Fig. 2) four times against 1 1 each. Dialyzed water and then lyophilized.

Weighing

Fraction 1 ₇₀ mg

Fraction 2 216 mg

Fraction 3 31 _5mg

(49.3% of theory)

310 mg of fraction 3 were dissolved in 3 ml of 1.5 M essential acid / 7 M urea / 0.05 M NaCl (pH 3.0) on a column (1.5 χ 45 cm) with SE-Sephadex inf carried, which equilibrates with the same buffer Was.

835 3-

¹ 10

The elution took place by means of a linear gradient of 250 ml start buffer and 250 ml feed buffer, which had the same composition as the dci start buffer, but was 0.2 M in NaCf. Tue « Eluate below the maximum (hatched part, see Fig. 3) was three times for 1 hour against I 1 dist Dialyzed water and lyophilized. The residue was purified by column chromatography (3 χ 60 cm) with Sephadex G-25 in 0.05 M ammonium bicarbonate solution of residual salt and urea freed and the eluate lyophilized.

Yield: 98.2 mg (15% of theory).

■ 278 = 5490 (in 0.05 M ammonium bicarbonate solution. pH 8.2) (see Fig. 4).

Paper electrophoresis at pH 2: uniform.

R _tns (electrophoretic mobility, based on insulin) = 0.74.

Free amino groups (dansyl chloride method according to W. R. Gray, Methods in Enzymology. Vol. 11.139 [1967]): Phenylalanine Oxalylinsulin crystallizes from citrate buffer containing zinc ions (.1. Schlichtkrull. Acta Chem. Scand. 10. 1455 [1956]) ir Form of small prisms.

Example 2
N ^lA1 .N ^cH29 -Succinylinsulin (bovine)

640 mg crystall. Bovine insulin was mixed with 43.2 mg (12OuMoI) of bis-p-nitrophenyl succinate within 3 hours implemented under the conditions described in Example 1. Reconditioning and gel chromatography were also carried out as described in Example 1.

Weighing

Fraction 1 90 mg

Fraction 2 105 mg

Fraction 3 411 _m o

(64.2% of the
Theory)

410 mg of fraction 3 were, as described in Example 1, chromatographed on SE-Sephadex and worked up. The main fraction (hatched, cf. rig. 3) gave 139 mg (21.7% of theory) succinylinsulin after the chromatography on Sephadex G-25.
... ' ^{2T | f = 554} ° (in 0.05 M ammonium bicarbonate solution. PH 8.2).

Paper electrophoresis at pH 2: Uniform.
Ki "= 0.77.

Free amino groups: phenylalanine.
K-nstallform: spherical particles.

Example 3
N »«, N ^tM9 -glutarylinsuIin (bovine)

/ iir, ^{40 g crystalline} ^ - ^Bark bis-p-nitrophenyl glutaric untei were rinsulin 44.8 mc ^ Μ υμΜοΙ) aen reacted described _in Example 1 Bedinguneen. Work-up and gel chromatography were also carried out as described in Example 1.

Weighing

54 mg

195 mg

330 _m g

(51.6% of the
Theory)

^{the fraction} '° n 3 were, as in Example 1 n, chromatoerated on SE-Sephadex and

worked up. After desalting by gel filtration to Sephadex G-25. followed by isoelectric repelling at pH 4.8. were from the main faction (cf. FIG. 2) 110.3 mg (17.3% of theory) gimarylin insulin receive.

I ₂ IH = 5400 (in 0.05 M ammonium bicarbonate solution).

Paper electrophoresis at pH 2: Uniform.

R _1n , = 0.74.

Free amino groups: phenylalanine.

Crystal shape. Matted little needles.

Example 4

N ^lA1 .N ^tll29 -Adipoylinsulin (beef)

ii) 1.27 g (200 μΜοΙ) crystall. Bovine insulin were in 140 ml of dimethyl sulfoxide in the presence of 0.3 ml of triethylamine with stirring at room temperature drop by drop with a solution of 93.2 mg (240 μΜοΙ) 2ο Adipic acid - bis - ρ - nitrophenyl ester in 5 ml of dimethyl sulfoxide offset. After standing at room temperature for 60 hours, the reaction solution became like Worked up described in Example 1. After GC on Sephadex G-50 fine in 10% acetic acid was obtained:

Fraction 1 304 mg

Fraction 2 335 mg

Parliamentary group 3 587 me

(46.2% of theory)

580 mg of fraction 3 were in two equal portions of SE-Sephadcx chromatographs, as in Example 1 described. The main fractions (cf. FIG. 3) were combined after the freeze-drying and desalted by gel filtration (see Example 1).

Yield: 294 mg (23% of theory) adipoyl insulin.

, _27f) = 5650 (in 0.05 M ammonium bicarbonate solution. PH 8.2).

Paper electrophoresis at pH 2: Uniform.

R _lns = 0.73.

Free amino group: phenylalanine.

Crystal shape: small needles and spherical particles.

b) To a solution of 120 mg (18.7 μΜοΙ) crystall. Bovine insulin in 15 ml of dimethyl sulfoxide and 30 μΐ triethylamine were added dropwise within 2 hours 7.8 mg (20 μΜοΙ) adipic acid - bis - ρ - nitrophenyl ester in 2.5 ml dimethyl sulfoxide with stirring. A further 7.8 mg of ester in 2.5 ml of dimethyl sulfoxide were then added dropwise over the course of one hour. The reaction solution was immediately treated with methanol / ether, the precipitated insulin derivative was washed with methanol / ether (1: 9) and briefly dried in vacuo. It was then dissolved in a mixture of 1 ml of glacial acetic acid and 9 ml of water and applied to a column (3 × 200 cm) with Sephadex G-50 fine in 10% acetic acid and chromatographed. The eluates (see FIG. 1) were dialyzed and lyophilized.

Weighing

Fraction 1 29 mg

Fraction 2 14 mg

Fraction 3 55 mg

(45.8% of the Theory)

Example 5
N ^lU , N '"" - adipoyl insulin (sheep)

To a solution of 320 mg of crystalline Sheep insulin (50 μΜοΙ) in 35 ml of dimethyl sulfoxide and 75 μΐ triethylamine was a solution of 23.3 mg (60 μΜοΙ) adipic acid bisp-nitrophenyl ester with stirring at room temperature added dropwise in 4 ml of dimethyl sulfoxide within 4 hours. The reaction mixture; stood for 48 hours at room temperature and was then worked up as indicated in Example 1. The crude product was chromatographed on Sephadex (column 3 × 200 cm) as in Example 1.

Weighing

Fraction 1 88 mg

Fraction 2 74 mg

Fraction 3 94 mg

(29.4% of theory)

Fraction 3 contained, according to quantitative paper electrophoresis, 59% N ^IAI , N '"" -adipoyl insulin

Example 6
N ^jA1 .N ^cB29 -Pimeloyl insulin (beef)

640 mg crystall. Bovine insulin were taken with 48.2 mc (120 μΜοΙ) pimelic acid-bis-p-nitrophenyl ester the conditions described in Example 1 implemented. After 44 hours the reaction mixture became worked up and the crude product chromatographies

Weighing

Fraction 1 194 mg

Fraction 2 Ulms

Fraction 3 288 mc

(45% of the
Theory)

As described in Example 1, 280 mg of fraction 3 were chromatographed on SE-Sephadex. The Product isolated from the main fraction (see FIG. 3) was desalted on Sephadex G-25.

Yield: 132 mg (20.6% of theory).

, -, - "= 5970 (in 0.05 M ammonium bicarbonate solution. pH 8.2).

Electrophoretic purity: 95%.

R, " = 0.73.

Free amino group: phenylalanine.

Crystal shape: matted needles.

Example 7
N ^lAI , N ^cB29 -Suberoyl insulin (beef)

640 mg crystall. Bovine insulin were reacted with 49.9 million pounds (12OuMoI) of suberic acid bis-p-nitrophenyl ester and worked up after 48 hours, as described in Example 1. After gel filtration the following was obtained:

Fraction 1 137 mg

Fraction 2 122 mg Fraction 3 331 mg

(5i, 7% of theory)

320 mg of fraction 3 were, as described in Example 1, chromatoerated on SE-SeDhadex. Tue <

Main fraction (see Fig. 3) was transferred to Sephadex G-25 Example K)

desalted, then 4.8 isoelectric pH environmentally N .N ^{JA1 l (i: 9} -Sebacoylinsulin (Sch wine ι

falls and lyophilized again. . ", ··, ·

Yield: 138.8 mg (21.6% of theory). 640 mg crystall. Black insulin was only 53.3 _my

i-, ₇₈ = 5110 (in 0.05 M ammonium bicarbonate- 5 (! 20 μΜοΙ) sebacic acid-bis-p-nilrophcnylesUr uni-

solution, pH 8 ~) uesetzt, as described in Example 1. After 2 ()

Electrophotic purity (pH 2): 98%. standing at room temperature was

^ _1n - o, 74. work.

Free amino groups: phenylalanine. Weighing out after gel filtration

: Crystal shape: Small needles. ^I0 fraction 1 190 m »

Fraction 2 139 mg

Fraction 3 280 mn

Example 8 (43.7% of

N ^7A1 .N ^{£ B29} -Azelaoylinsulin (bovine) _{| 5} theory) 640 mg crystall. Bovine insulin with 51.6 mg "fraction 3 contained according to the quantitative elckim- (120 _: λΜο1) azelaic acid c-bis-p-nitrophenyl ester umphorctic analysis 70%, Sebacoyhnsuhn. Set, as described in Example 1. After the gel filtration (see F i g. 2) were obtained: Example 11

20 N ^lA! .N '" ² '^< - (Bis-NN - ten.-butylo \> earbon \ li-

Fraction 1 166 mg cystinylinsulin (bovine) "

Fraction 2 121mg

Fraction 3 311 mn a) To a solution of 640 mg of crystall. Bovine insulin

(48% of that in 75 ml of dimethyl sulfoxide and 150 μΙ 1 riätlnlamin

Theory) J5 became a solution with stirring at room temperature of 95.8 mg (12 () μΜο1) N.N'-Bis-teri.-hutvl-

300 mg of fraction 3 were on SE-Sephadex oxycarbonyl-cystine-2.4.5-trichlorophenyl ester in 5 ml

Ehromatoüraphiert. as described in Example 1. Dimethyl sulfoxide within - «hours urter

The work-up of the main fraction (possibly Fi ».3) stirring dropwise. After standing for 22 hours, the

From after desalting 134.3 mg azefaoyl insulin 30 reaction mixture, as described in Example 1.

Γ21% of theory). "Worked up The crude product was sent to Sephadev

; _27S = 6060 (in 0.05 M ammonium bicarbonate G-50 in 10%, acetic acid chromatography

solution). / Uiswaaticn E 'electrophoretic purity (pH 2): 94' '.

R _1n = 0.77. ί5 fraction al 191 mg

Free amino groups: phenylalanine. Heration a 2 '- "- mg

Crystal shape: small needles. Fraction a3 261 mg

(40.S ",, of theory 1 B e i s ρ i e I 9 40

μίλι MIH29 c u ι · ι-, n · 1 b) The reaction was under the same Hedinnun-jen

N ' ^AI .N ^tBM -Sebacoylinsulin (beef) _{wjc jm Bcispjc} , _{1 | a) gcführ} , _{jcdodl umcr χ} ^ _η _

640 mg crystall. Bovine insulin were made with 53.3 mg of 110 μΐ N-methylmorpholine as a base.

(120μΜο1) sebacic acid-bis-p-nitrophcnylestcr um-.

■ · u ■ r> · · 1 1 u 1. ■ u au u 1 Select set as described in Example I. Deviating 45

worked up so that the reaction pro fraction b I 206 mg

product after dialysis by freeze-drying iso-fraction b2 159 mg

was licrt. The crude product was in 4 m! Glacial acetic acid fraction b3 272 mg

and 16ml of water and dissolved as in Example I (42.5 "., der

Chromatographies. 50 I theoryi

Weighing out 250 mg of fraction a 3 was as in Example 1

Fraction 1 175 mg. separated at SE-Sephadex. The main

; ■ Fraction 2 143 mg fraction was determined by chromatography on Sepha-

: Fraction 3 278 mg 5s dex G-25 desalted.

f (43% of the yield: 103.6 mg (16.5% of theory ».

Theory): _27H = 5400 (in 0.05 M ammonium hicarhonate

£ solution. pH 8.2).

J 270 mg of fraction 3 were, as in Example 1, paper electrophoresis (at pH 2): Uniform.

<:. described, at SE-Scphadex Chromatographien, the ho R _in , - 0.77.

| _μ Main product (cf. FIG. 3) was transferred to Sephadex Free amino groups: phenylalanine.

§3 G-25 desalinated. In the same way, 260 mg of Iraq

Yield: 121.2 mg (19 "" of theory). Lion h.V 91.8 mg (14.3% of the theory iBis-hui \ lox \ -

Ii ^; 27k = 5500. caronyl-cystinyl-insulm obtained.

Paper electrophoresis at pH 2: Uniform «,. <; .-- "= 5900. '

^ ;, i \ = 0.78. Paper electrophoresis (at pH 2): uniform.

Free amino groups: phenylalanine. R _1n , ■■ - 0.70.

Crystal shape: small needles. I amine group: phenylalanine

Example 12 N ^1A1 , N ^tB29 -Cystinyl-insulin (bovine)

40 mg N ^aA \ N ^tB29 - (B ^; s-butyloxycarbonyl) -cystinylinsulin (Example 11) were _{dried in vacuo over P 2} O ₅ and KOH for 15 hours. 0.3 ml of trifluoroacetic acid was then added in the centrifuge tube and the solution was kept at room temperature for 1 hour. The protein was then precipitated with 10 ml of absolute ether, the residue was centrifuged off and washed several times with ether.

Yield after drying in vacuo over Ku ₅ and K OH: 42 mg (about 96% of theory).

'278 ⁼ 5560 (in 0.05 M ammonium bicarbonate solution).

Electrophoretic purity (at pH 2): Uniform.

however, both buffer solutions were 700 ml each. The work-up of the main fraction (see FIG. 3) gave 139 mg of undecanedioyl insulin after desalting (22.5% of theory).

Paper electrophoresis at pH 2: Uniform.

^f 27b = 5390 (in 0.05 M ammonium bicarbonate solution, pH 8.2).

R _lns = 0.74.

Free amino groups: phenylalanine.

Crystal form: spherical particles.

Example 15 Example 13

Representation of N ^lA1 .N ^{£ lJ29} - (N-Benzyloxycarbonylj-glutamyl-insulin (bovine)

To a solution of 640 mg of crystalline. Beef insulin in 75 ml of dimethyl sulfoxide and ΙΙΟμΙ N-methylmorpholine a solution of 62.7 mg was obtained within 5 hours with stirring at room temperature (120 μΜοΙ) N - benzyloxycarbonyl - glutamic acid «, - / - bis-p-nitrophenyl ester added dropwise. After 22 hours Standing was worked up as in Example 1, followed by chromatography.

Weighing

Fraction 1 106 mg

Fraction 2 129 mg

Fraction 3 257 mg

(40.2% of theory)

N ^lA1 , N ^tB29 -dodecanedioyl insulin (beef)

640 mg crystall. Rinderinsuün were with 56.7 mg (120 μΜοΙ) dodecanedioic acid - bis - ρ - nitrophenyl ester implemented as described in Example 1, but the reaction solution was 18 hours after addition of the ester worked up. After the gel filtration, the following were obtained:

As described in Example 1, 250 mg of fraction 3 were chromatographed on SE-Sephadex. After desalting by chromatography on Sephadex G-25 the following was obtained: 105 mg (16.4% of the Theory).

Electrophoretic purity (pH 2): 95%.

Ai "= 0.76.

Free amino groups: phenylalanine.

Example 14 N » ^AI , N ^{cB2 <l} -undecanedioyl insulin (beef)

640 mg crystall. Bovine insulin were given with 55.0 mg (120 μΜοΙ) undecanedioic acid bis-ρ-nitrophenyl ester implemented as described in Example 1, but the reaction solution was already 18 hours after Addition of the ester worked up. After the gel filtration (see Fig. 2) the following were obtained:

Fraction 1 144 mg

Fraction 2 117mg

Fraction 3 311 mg

(48.6% of theory)

300 mg of fraction 3 were on a column (2.7 χ 40 cm) with SP-Scphadex chromatography, as described in Example 1, the volumes of

Fraction 1 140 mg

Fraction 2 157 mg

Fraction 3 276 mg

(43.1% of
Theory)

260 mg of fraction 3 were on a column (2.7 χ 40 cm) with SP-Sephadex chromatography, as described in Example 1, but the buffer volumes were 700 ml in each case. Work-up the main fraction (see FIG. 3) gave after desalting 108 mg (17.9% of theory) dodecanedioyl insulin.

, _{lls =} 5870 (in 0.05 M ammonium bicarbonal solution. pH 8.2).

Paper electrophoresis (at pH 2): Uniform.

R _1n = 0.73.

Free amino groups: phenylalanine.

Crystal form: spherical particles.

Example 16
_n * ai N ^tB29 -Tridecanedioylinsulin (beef)

640 mg crystall. Bovine insulin were made with 58.4 mg (120 μΜοΙ) tridecanedioic acid - bis - ρ - nitrophenyl ester implemented as described in Example 1, but the reaction solution was 18 hours after addition of the ester worked up. After the gel filtration, the following were obtained:

Fraction 1 143 mg

Fraction 2 154 mg

Fraction 3 297 mg

(46.4%)

290 mg of fraction 3 were on a column (2.7 χ 40 cm) with SP-Sephadex chromatography, as described in Example 1, but the buffer volumes were 700 ml in each case. Work-up the main fraction (see FIG. 3) gave, after desalting, 109 mg (17.4% of theory) of tridccanedioylinsulin.

, _27g = 5880 (in 0.05 M ammonium bicarbonate solution, pH 8.2).

Paper electrophoresis (at pH 2): Uniform.

*, "" = 0.75.

Free amino groups: phenylalanine

Crystal shape. Spherical particles.

For this purpose 2 sheets of drawings

Claims (1)

Claims: 22 1. Bifunctionally crosslinked insulin derivatives in which the <i-amino group of glycine ^{At of} the Α chain with the i-amino group of lysine " ^{29 of} the B chain through a dicarboxylic acid bridge of the formula - C - X - C- fll