DE2803238A1 - SYNTHETIC PEPTIDES - Google Patents

Description

The present invention relates to therapeutic useful peptides, their derivatives and acidic and basic salts of these peptides and peptide derivatives.

The therapeutically useful peptides according to the invention contain at least 4 amino acid units and can contain 12, 13 or more of these units or groups.

The inventive products are good therapeutic products Middle; because they have the ability to have smooth muscles contract] !, Histöiuin from "Mar.t" -waves ireizusebzoii ι; · ιά clic

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BATH ORIGINAL

Improve vascular permeability. Certain according to the invention As explained further below, connections are therefore suitable, because they have the opposite effect. In other words: some peptides according to the invention are agonists or promoters, on the other hand, antagonists or inhibitors are different. Certain compounds can be used to treat cardiac arrhythmia will.

The carboxyl terminus of the peptide is an ainidino-substituted one δ <-amino acid, such as arginine or canavanine. Of simplicity it is referred to as arginine in the description below for the sake of it. The arginine is in a peptide bond via its c <-amino group to the carboxyl group of a lower one aliphatic amino acid bound, which expediently up to contains 4 amino acids. The presently preferred second amino acids are glycine and alanine.

The third amino acid of the peptide is linked to the amino group of the second amino acid via its carboxyl group. she is a hydrophobic amino acid such as s, B. Isoleucine, Leucine »Valine, Methionine, Phenylalanine, Tyrosine or Tryptophan, Leucine will Currently preferred because it is in both D and L shapes readily available and contributes satisfactorily to the effectiveness of the peptide.

The identity of the fourth amino acid does not seem to be crucial; To achieve good effectiveness, a fourth amino acid should be present. Practically suitable

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Each of the twenty noraalerv / oiae occurring amino acids. Compounds with particularly good or potency are obtained when the fourth amino acid eats glycine or glutamine. However, other amino acids such as alanine, asparagine, serine, tyrosine or phenylalanine can also be used as well.

Due to the presence of a fifth, hydrophobic amino acid, like leucine, the effectiveness of the peptide is significantly increased. Other hydrophobic amino acids such as those above mentioned are also useful.

It was found that the WirksaTake.it increased with Korider the peptide chain has up to 13 amino acids. Bio through this However, they do not justify the massive improvements achieved. the cost of producing polypeptides with a a significantly higher number of amino acid units are formed.

The compounds according to the invention, the first amino acid of which is arginine, are agonists. Is the arginine about his Carboxyl group attached to the amino group of a lower aliphatic amino acid such as glycine, proline, alanine or serine, thus antagonists are obtained as products. At low Dosages they inhibit the contraction of. smooth muscles, the release of histamine and the vascular permeability, The amino axes to which the arginine is bound can therefore can be referred to as the inhibiting amino acid. The currently preferred inhibiting amino acid is glycine, because it is is readily available and can be added to the molecule without introducing a new asymmetric center,

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The effectiveness of the peptides according to the invention can also be modified in other ways, for example by using a D-amino acid instead of a naturally occurring L-amino acid as a component of the peptide. Various products in which! ■ -arginine has been replaced by D-arginine oppose the hydrolytic effect of Carboxypeptidar.eB. The product obtained is therefore stable for a long time and can be used for long-term preparation of preparations. ^r jr \ k can be used. The same result can be achieved by substituting proline in the second position.

Due to their ^one amphoteric nature, the erf indungsgemäa.i <can ett products in Fon "pharmaceutically acceptable SALR-se v applied; ground that both metal salts v / he also may be acid addition salt-G. These salts have the advantage of being soluble in water and are not particularly suitable for parenteral use. The metallic salts, in particular: the alkali metal salts, are relatively stable and are therefore often preferred to the acid addition salts. Sodium salts are particularly preferred because they are easy to prepare.

For the preparation of the acid addition salts according to the invention acids that contain non-toxic anions are used, e.g. hydrochloric, sulfuric, phosphoric, acetic, lactic, lemon, tartaric, oxalic, succinic, maleic, gluconic, sugar acids or the like. A large number of non-toxic derivatives of the polypeptides according to the invention are suitable. The products, especially those inhibitors that have a glycine carboxyl

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Terminal aufwc-isen _f eg can with thionyl chloride into the acid chloride U ^;! Be eweiidelt which subsequently with ammonia to ac-rn amide ucgesetst. Amines naturally form N-substituted aides.

Other useful derivatives are obtained by using the Modified free functional groups on the peptide main chain. So can. 8.B. with free hydroxyl groups and free Amino groups can easily be formed into derivatives. The KIaκso of derivatives in which a free hydroxyl group, which can be either aliphatic or aiOE-atic, with a Alkanoyl or alkonoyl group has been esterified to the 18 carbon contains atoms or more. You can still get a free one Acylate amino groups with alkanoyl or alkenoyl groups, which contain up to about 18 carbon atoms. In both In cases, preference is given to the derivatives in which the derivative-forming Group contain 11 to 18 carbon atoms, because the longer ones. Hydrocarbon chains increase lipoid solubility of the molecules and improve their transport across the cell boundaries.

The erfindungsgeinässen compounds can by means of many. Various processes can be prepared for the synthesis of simple and complex polypeptides or even of relatively low molecular weight proteins are known. Generally, these procedures involve an step-by-step synthesis, irideia gradually the individual amino acids added and increasingly larger molecules formed

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will «The amino acids are produced by condensation between the carboxyl group of an amino acid? and the Aiuinop.ruppe one other amino acid linked together, forming a peptide bond arises. To regulate these reactions, one jsusg the amino group of one acid and the carboxyl group of the block others. The blocking groups should be chosen so that they can be removed without harming the polypeptides The resulting peptide bonds can be easily removed again by racemization or hydrolysis. Some amino acids contain additional functional ^ groups, 2.B. the hydroxyl group "of tyrosine or threonine. In general, too these additional groups with an easily removable Blocking & medium blocking so that you get the desired Do not interfere with condensation to form the peptide bond.

There are already many methods of producing polypeptides and numerous blocking centers3. known. Most these processes are also suitable for the process according to the invention Class of polypeptides. Suitable procedures are shown in the examples below.

A process which is currently particularly preferred for the preparation of the compound according to the invention is the Merrifield process. Here, an amino acid is bonded to a resin particle by an ester bond and the peptide is gradually formed by protected amino acids are gradually added to the growing chain. This procedure is well known and is being used

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described in many publications; see e.g. Erickson and Morrif, icld (1976), Solid-rhaae Peptide Synthesis, "The Proteins ", Neurath and Hill Aoadraic Pro kg, New York, pp 255-527.

For the sake of simplicity in the present description the usual abbreviations used for the various amino acids. These abbreviations of the The most important amino acids according to the invention are:

Arginine Arg Alanine AIa Leucine - Leu Glycine - GIy Histidine His Serine Ser Glutamine Gin

As already stated, the agonists according to the invention intensify the contraction of smooth teats and the release of histamine and improve the vascular permeability. They are therapeutically useful to induce inflammatory responses in mammals in which this response is impaired. So they can _t be used to help your body build its defenses against it!

Penetration of infectious microorganisms or other bej

mobilize loads, antagonists are clinically useful bar at local or systemic, episodic or traumatic Muscle contraction, hemamine release, or increased j

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Vascular permeability. For example, they can be found in nasal sprays Asthma attacks or bronchial allergy. Ausserdeni are they Prophyl & ktika for such syndromes as κ.B. the shock associated with dengue fiebor.

The products of the invention are useful therapeutic Means for mammals or humans and show good stimulating and inhibiting properties even at very low doses Effect. Your doctor or veterinarian can determine the most suitable dosage for your intended use. It can vary from patient to patient and depends on the size of the patient, the disorder to be treated and others, factors known to those skilled in the art. The products can be administered in very high doses, e.g. up to 2 g or re.thr per day, in general they are given in dosage units & n manufactured that contain up to about 250 mg of the active ingredient. The number of units to be taken per day can be prescribed by the doctor according to the disorder to be treated.

The products can be administered alone; however, in general, they will be admixed with pharmaceutically acceptable, non-toxic carriers. The proportion of carrier material depends on the usefulness and the chemical nature of the carrier, the chosen mode of administration and the usual pharmaceuticals Practices. For intravenous or intramuscular administration, the products are processed into a sterile solution, which contains other solutes, see B. a sufficient amount of saline or glucocs, go that one

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j :. otL'i'iJ.ijouo Li5;.:. ung is obtained. For external use icüiiiioü the frcdulcte are mixed with suitable carriers, κ. B. with Vaöolin or other inert, semi-viscous materials. Dosing units with an intestinal * coating can are made and contain as tablets or capsules such carriers as starch, lactose, certain types of clay and the like.

Although arginine is the currently preferred first amino acid, other (Λ -amino acids, such as canavanine, can also be used -iordon. All can be viewed as arginine analogs ■ ", / orden, f '; -. Alo a .0. Amidino group ^ CC = NH) -ITIl _2- / contain. • '■ He can ii a larger or smaller Ansalil ^ ^r on Methylsn- ■:, r) .or ImiiiOfiruppen in dor, connected with the C \ -carbon atom π fi: 5l) f. ^> rJ.:--tte auiwoison. One or more of these groups izöanen even with another group, such as an oxy, thio, inino or carboxyl group, orsetst v / ground. One or more of the n.lt carbon or nitrogen-linked hydrogen atoms kt ^; r .-; v: -n be linked by its alkyl group, which typically contains up to 6 carbon atoms, but preferably a methyl or ethyl group as mentioned above , the acid can be in D- or L-form.

The following examples illustrate the present invention.

* "entericaily ¹¹

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Example 11 Production of L-AH? Vi: yl ~ L-sery] -T-hi nt j clyl-L-

Preparation of N ^ -tert.-Batyloxycurbonyl-ii '-''- (4-toluenesulfonyl) ~ L-arginyl-oxymethyl- (styrene-copoly-1 ^ -divinylbinsol) (II): A solution of 1.3 g of N ^ot , -tert.-Butyloxycarbünyl ~ N ^ - (4-toluenesulfonyl) -L-arginine in 15 ecm ethanol and 1.35 ecm water was brought to a pH of 7.1 by adding a solution of 0.43 g caesiuin bicarbonate in 1.35 ecm water added v.ixrde. The solution thus obtained was evaporated to dryness at 40 ° C. under reduced pressure. Then 25 ecm of benzene was added and the mixture was again evaporated to dryness under the same conditions; this process was repeated four times. The solid thus obtained was 2.5 hours at a pressure of 1 Torr and gave 1.7 g of the cesium salt of Boc-Arg (Tos) (i) in the form of a white solid. benzene) (0.23 millimoles per gram of polystyrene) swell in 15 ecm of N, N-dimethylformamide (DMF) and mixed it with a solution of 1.7 g of salt I in 10 ecm of DMF. The mixture was mechanically stirred and stirred for 75 hours held 5O ⁰ C. Then a solution of 3.7 g of salt I in 25 cc of DMF was added and the mixture was stirred for 95 hours and maintained at 50 ⁰ C. the resin was collected on a sintered glass funnel and washed successively Rait DMF (three times 25 ecm), absolute ethanol (three times 25 ecm) and dichloromet han (three times 25 ecm each) washed. After drying at 1 torr for 2 hours, Resin II (3.3 g) contained 0.17 millimoles of arginine per gram of polystyrene; This was determined by 10 nig portions 30 minutes at 0 ⁰ C with

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Licho material for 24 hours at 110 ⁰ C with 6 η HCl hydrolyzed and exposing the hydrolyzate of an amino acid analysis; 10 i> anisole treated in liquid IJF, the acid loei.

Production of N -tert.-Eutyloxycarbonyl-L-alanyl-O ^ -benzyl "L-seryl» N ^iKl - (4-toluenesulfonyl) -L-histidyl-L-leucyl-glycyl-L-leucyl-L-alanyl- N "~ (4-toluenesulphonyl) -L-argir> y 1-oxyme Lh / 1- (styrene-copoly-1 ^c X-diYinylbenzene) (III): One liCGS 2.0 g of Boc-Arg (Tos) - Resin II (0.34 millimoles arginine) in a 75 cc;> glass reaction vessel, which carried a sintered glass disk, swell in 40 ecm dichloromethane. By pressurizing the vessel with air or nitrogen and / or using reduced pressure at the outlet below the sintered glass pane, the resin was filtered until most of the solution had been removed.

A. The process step for removing the protective groups was carried out by adding 40 ecm of a 1: 1 solution (v / v) of trifluoroacetic acid and dichloromethane to the vessel, stirring the mixture for 1 minute and filtering in the manner described above. A further 40 ecm of the 1: 1 solution of trifluoroacetic acid and dichloromethane were then added, and the mixture was again stirred for 30 minutes and filtered.

B. The washout step was carried out in a similar manner by the resin successively with dichloromethane (five times 1 minute with 40 ecm each), 2-propanol (twice 1 minute with 40 ecm each) and Washed dichloromethane (five times for 1 minute at 40 ecm each time).

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C. In the neutralization stage the Harji was: old a 1:19 solution (Vol./YoI.) Of Ethyldiinopropylarcin and Dichlo? ⁿ ~ methane (three times each 2 Kjnuten luit ccr 40-i).

D. Washout step B was repeated.

E. The coupling stage took place by adding the J for κ 10 ccia a dichloro-ethane solution of (rewUiiGchtGii Boc - /> - iaj.'ocäur * e (3 millimoles per milliniol of the original Argynin) add'.t.n, 2 minutes, add 10 ccia of a DichlorKctbi-r - solution of NjN'-dicyclohexylcarbodiinide (3 milliKol pi ^ o M.i3. "i.i ~ laol the; original arginine), remchte for another 30 minutes and filtered afterwards.

F. Ausv.'abchstufe 13 was repeated.

G. The neutralization stage C is repeated. H. The ash level B is repeated «

I. The coupling stage E was repeated. J. Washout B was repeated.

Steps A to J form a complete synthesis cycle, in the course of which a protected amine acid residue is added to the peptide is added. This cycle was first made with Boc ~ L-alanine and then one after the other with Boc-L-Leucine, Boc-Glycine, Boc-L-

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BATH ORIGINAL

Leucine, Boc-N ^m - (4-toluene-ulionyl) -L-histidine, Boc-0 -benzyl-L-serine and Boc-L-alanine by Ulirt; "Boc" stands for N -tert-butyloxycarbonyl in each case.

Removal of protective groups and cleaning of the L-Alanyl-L-seryl-L-histidyl-Jf-leucyl-glycyl-L-leucyl-L-alanyl-I ^ ai ^ ginins' (IV): 0.26 g of the protected octapeptide resin III treated for 1 hour at ⁰ ° C. with 10 pounds of anisole in 6 ecm of liquid HF. After evaporation of the HF with a water jet pump and then with a vacuum pipette, the resin was washed with ether (three times, 2 com) to remove anisole, and finally with trifluoroacetic acid (five times, 2 ecm each) to remove peptide IV . After evaporation of the trifluoroacetic acid, the residue was dissolved in 5% aqueous acetic acid and lyophilized, among other things to provide the peptide; this was dissolved in 2 ecm 1% aqueous acetic acid and filtered in a 2.5 cm: -: 100 cm column with Bio-Gel P-2 (100-200 mesh, Bio Rad), through the 1% acetic acid at one speed of 23 ccra / h was pumped. The quantities of the ninhydrin-positive main MaxiiQum (175 to 300 ecm) were combined and lyophilized. Then 19 mg of the residue in 0.01 cm NaCl, 0.01 M NaH ^ PO. (pH 4.5-1.5 ecm) dissolved and placed in a 1 cm x 50 cm column with carboxymethyl cellulose (Cellex CM, Bio-Rad), which at 30 ccin / h and a linear salt gradient of 0, 01 to 0.20 m NaCl was operated (both solutions 100 ecm, 0.01 m HaH ,, P0 <, pH 4.5). The areas of the main peptide inaxiwa (130 to 170 ecm) determined by UV absorption, at? 06 nr, were mixed together and lyophilized. The remaining mixture

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from salt and peptide was dissolved in 2 ecm 1% acetic acid and desalted in a 1.5 cm χ 85 cm column with Bio-Gel P-2, which worked with 1% acetic acid at 15 ccm / h. The amounts of the main peptide trauma (50 to 75 ecm) determined with an absorption of 206 mg were extracted and lyophilized and yielded the pure octapeptide IV (10 mg). This material gave the expected amino acid analysis (see Table I) and amino acid sequence as determined by automatic Edaan degradation. In addition, it showed a simple ninhydrin-positive and Pauly-positive point on thin-layer chromatography (R ^ 0.73 versus valine with R. * 0.64) in a solution with 15: 12: 10: 3 (v / v) 1-butanol, water, pyridine and acetic acid xxad in high-voltage electrophoresis ( ^R Arg 0 _? 76 at pH 5.0 ^ ^R Arg °> ^{5 & at} P ^{H 6} ^ * ^{where R} Arg is the distance between the middle points of the peptide IV - and is the valine stellon divided by the distance between the centers of the arginine and valine sites).

All peptides listed in Tables I and II were practically ^r in the same V. else made "

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Table I.

away

Composition of amino acids, col.-ratio, tie activity

No. Followed the Amino Acids ' His Arg Ser Gin GIg Ala leu J ^ R ^ A (nl'ol)

1 Gly-Leu-Ala-Arg 0 1.04 0 0 1.08 10.9 1.00 0.73 1200-1400

2 Leu-Giy-Leu-Ala-Arg 0 0.8? 0 0 1.12 1.12 2.00 0, SO 150-300

3 His-Leu-Gly-leu-Ala-Arg 0.98 0.97 0 0 1.18 1.09 1.00 0.79 10-20 α, 4 Ser-His-Leu-Gly-Leu-Ala- Arg 0.39 1 _r 00 0.93 0 1.06 1.05 2.00 0.77 6-10

S 5 Ala-Ser-His-Leu-Gly-Leu-

S Ala-Arg 0.95 1.00 O ₅ 97 0 1.13 1.94 1.94 0.73 5-8

^ω 6 CrUCO-Ala-Ser-His-Leu-Gly-

^ ² Leu-Ala-Arg 0.90 1.00 0.93 "0 1.32 2.00 2.33 0.81 G-'2

° 7 Arg-Gln-His-Ala-Arg-Ala-Ser-

^ His-Leu-Gly-Leu-Ala-Arg 1.97 3.00 1.04 1.10 1.11 3.30 2.18 0.71 4-7

c "8 Ala-Ser-His-Leu-Gly-Leu-

Ala-Arg-Gly 0.90 1.00 0.97 0 2.20 1.92 2.09 0.73 280-560

9 Arg-Gln-His-Ala-Arg-AIa-Ser- · -
His-Leu-Gly-Leu-Ala-

Arg-Gly 1.90 3.00 O _? 94 0.96 2.30 3.09 2.20 0.71 240-360

Ratio of peptide nobility to migration of the ^ ₀ riorcn Randos dec solvent on silica gel-Dilonschicht-Platteri in 15: 12; 1C; 3-.uos'.ir-, s (vol.) Of 1-Buxars.ol, '» vase he, pyridine and ^ ra acetic acid. _O

_G Kens © an peptide per 10 com Tyrode's solution, un strip ej, noa Meerschvieincheii-Ilsuris roll- p ^ 2 to contract constantly. <x>

lO CO

TabaIle_II

Composition of the thin-layer values ileum, amino acids _T Hol.-Rat. (R _f χ 100) activity ⁰

No. Sequence of the amino acids GIv Leu AIa Arg __A__ _3 ^ C ^ Ό_ _ (r "ol)

10 Ala-Ala-Ala-Leu-Gly-Leu- ■

Ala-Arg 1.03 2.00 4.04 1, OS 16 45 56-6-3

11 AIa-Ala-Leu-Gly-Leu-AIa-Arg 1.02 2.00 3.06 1.08 16 45 60 - 10-14

12 Ale-Leu-Gly-Leu-Ala-Arg 1.03 2.00 1.99 1.05 22 50 65 - 5-11

13 Asn ~ Lys-i? Ro-Leu-Gly-Leii "-Ala- -,

Arg 1.03 2.00 1.01 1.03-17 30-14-27

H CH-CO-ieu-Gly-Leu- AIa- Arg 1.03 2.00 1.02 1.05 - 66 66 - 36-45

¹ 5 CH ^ CO-His-Leu-Gly-Leu-Ala-Arg 1.05 2.00 1.01 1.03 ° 63 43 13 - S-1?

^ 16 CH ^ CO-Ala-Leu-Gly-Leu-AIa-Arg 0 _? «3 2.00 1, SS 0.91 42 - - 13 1S-3S

· * +. 17 CH-CO-Ala-Leu-Gly-Lsu-Ala-Can® 0.99 2.00 1.? 8 45 - - 21 85-170

18 CH ₇ CO-Ala-Ala-Ala-Leu-Gly-

oo ^J Leu-Ala-Arg 1.21 2.00 4.27 1.02 39 - - 10 7-13

Ratio of peptide mobility cur Vco.derung d? S anterior. Edge of the Lccunfvc ^ ittels on

"■■ Kenge of peptide per 10 ccsi Tyrods's solution, un Streif er :, one.:. !!" ■■ 2rr.c: veir; che: '; -. I.lD; r. "; 3 complete l'U contract.

> ^c Also; His - 0.96 r °

§ ^ Also: Asn - 1.00; Lys - 1.02; ? ro -1.01 ^

S ^e L-Canc-, Tanin (Can) has a chain / T; H _? -C: -L.-O-I ': HC (= NH) -NH ₂ _7 _i 6 that of arginine (Ar§JJ

Claims (1)

1. Pt ptltt! - ,. it vci ') .. i.gi; tei). £.'> 4 IminoίΛχιτen _t characterized by the presence of a yiaidino-substituted C & ~ amino acid :; as the first amino acid at the carboxy! - end group -j v / oboi the first amino acid with a peptide bond ^: via its aminogi'uppa en the carboxyl group of a ri \. 'üiten Au-iiAO ^ LIurr. from the group: Glycine and alanine is bound; the av / eite auinotic acid is bound to a peptide bond via its am ir. ο group a /: clio carboxyl group evu.er third hydr-c -.- ht .- 'non amino acid; the civitte amino acid n: with a peptide bond VJdqt its are ino group to the carbo:;. ylg.rupi, .O a fourth aminob & nrö from the group: glycine, glutamine, alanine, asparagine, serine, Tyrc- Gin and phenylalanine is gebmi'ion; as well as derivatives of this peptide and the phariaaseatically useful acid addition salae and basic salts thereof. 2. Peptide according to claim 1, characterized in that the amidino-substituted C-amino acid is arginine. 3. Peptide according to claim 1-2, characterized in that the fifth amino acid is hydrophobic. bad original 809831/0785 original inspected - -vo - 4. Peptide according to claims 1-3, characterized in that the pvwoite Ar.üiKcäure alanine and the fourth amino acid glycine is. 5. Pep bid according to claims 1-3, characterized in that ßcn.'ohi the - //. Wlta as well as the fourth are i no acid is glycine. 6. Peptide η rieh claims 1-3, characterized in that the ΡΛ, ΊλΙΐΘ A ^T mjo. '; Acid is glyein and the fourth Aiidnosl'uvü is glutamine. 7. Peptide according to claims 1-3, characterized .. that the sv / eite Ainim-i-.aure .A3 en in and the fourth amino acid glutamine is. 8. Peptide according to claim 2-7 »characterized in that the Arginine is D-arginine. 9. Gly-Ieu-AIa-Arg. 10. Leu-Gly- Leu-AIa-Arg. 11 . His-Leu-Gly-Leu-AIa-Arg . 12 ♦ Ser-His-Leu-Gly-Ieu-Ala-Arg. 809831/0785 ßAD ORIGINAL 13. / ila-Ser-Jii ri ~ Leu - <n v-I-vu-Al ;: -Arf.:. H. CIi ₇ CO-Al; -Cer-Hin-I ^ ii-Gly-Jou-Ala-Ar ;;, 15. Arg-Gln-His-Ala-Ari ^ / aa-S'.r- -His-Leu- Oly-T-eu-Ala-Arc 16. peptide nacl; Claim 1 - O ₅ characterized [-, ekpnnZci paves that the ai3idino ~ f-ul; stituicrto C ^ -A: 'i ^') i.äuro nit a feptide-BinOiiu. ;; about their carboxy] i ^r ruppc an ede Amivo ^ L-iqyga vj.nuc Auinosliure au ;; Oov groupί CJyOrI ₁ P: coli) i, Älan.ijj and Sei'ij: bound wrong « 17, Ala-Sor-l-IiK · LtU-GIy-LvU-Air-Ar- ~ GIy. Of the 8098 3 1/0785