IL31992A - Synthetic thyrocalcitonins and method of making them - Google Patents

Description

SYNTHETIC THYROCALCITONINS AND METHOD OF MAKING THEM !U3ni> Thyrocalcitonins, which are polypeptide types of hormones, are present in the thyroids of mammals, including pigs and human beings, and act as serum calcium lowering agents by inhibiting bone resorption. Thyrocalcitonins have been isolated from mammalian thyroid, and a structure has been determined, see for example, Calcitonin « "Purification and Structure of Porcine ealei4>onj4n-l" by P. H. Bell, et al., Journal of the American Chemical Society/90:10/May 8, 1968. It should be noted that the exact structure is almost certainly not identical with that of the thyrocalcltonin in any particular mammalian thyroid as the processes of isolating it involve procedures which can change the structure somewhat. It is also likely that when thyrocalcltonin of the structure of the Barg and Bell application is administered to human beings for therapeutic treatment, reactions in the patient's body change slightly the exact chemical formula. Other workers have isolated thyrocalcitonins from mammalian sources . Typical of this work are the following: Tenenhouse, Arnaud, and Rasmussen, Proceedings of the National Academy of Sciences, Volume 5 pages 8l8-822 (1965); Hirsch, P.F., Gauthier, G.F., and Munson, P. L., Endocrinology, Volume 73* pages 244-252 (1963); Foster, G.V., Baghdiantz, A., Kunar, M.A., Slack, E . , Soliman, A.A., and Maclntyre, I., Nature, Volume 202, pages 1303-1305 (1964); Maclntyre, I., and Parsons, J.A., Journal of Physiology (London), Volume 183, pages 31-33 (1965); Maclntyre, I., Parsons, J.A., and Robinson, C.J., Journal of Physiology (London), Volume 191* pages 393-405 (1967); Kahnt, F.W., Riniker, B., Maclntyre, I., and Neher, R., Helvetica Chimlca Acta, Volume 51, Fascicle First, pages 214-217 (1968); and Franz, J., Rosenthaler, J., Zehnder, K., Doepfner, W., Huguenin, R., and St.

Guttmann, ibidem, pages 218-220.

This invention relates to new synthetic thyro-calcitonin active materials which have a shorter chain length than the full 32 amino acid compounds of the Barg and Bell application. It has been found that the shortest chain of amino acids, starting from the amino end, that has been proven to show thyrocalcitonin activity is of 16 amino acids . The activity is lower than that of thyrocalcitonin itself. The invention also includes further synthetic thyrocalcitonins with amino acid chains of from 17 to 31. The structures of the com- -pounds of this invention may be represented as follows: j Asn I •Cyc.Ser.Asx·Leu.Ser. Th .Cye.Val.Leu. Ser.Ala. yr. r . 3 wherein the terminal group of said polypeptide is a croup completing an amino group or a functional derivative thereof, the 0 terminal of said polypeptide is a group completing a carboxyl group or a functional derivative thereof and each -S. represents a conventional mercapto blocking group, or whon taken together represent an linkage and subscripts 1 through z, inclusive are aero or 1, with the provieo that when a subscript ie aero, the or each subsequent subscript in the anlno acid sequence is also zero, whereby the amino acid sequence of the polypeptide io preserved. The dote conventionally reprccont bondo connecting anino acids or bonds attaching groups thereon. As a typical example a dot at the C terminal or N terminal indicates that caid terminals may bo free (carboxyl or amino) or substituted. In cases of the latter,. ny of the usual substitutions may bo intended. Thus a C terminal dot may stand for a free carboxyl group or for carboxaaid© or for an eetorified carboxylic acid group or for another usual modified form. An N terminal dot is intended to be appropriately and similarly liberally interpreted to ctand for a free amino group, an acyla od amino group, or otherwise substituted amino group. " . dots abovo the sulfur atoao in amino acids 1 and 7 aay bo Joined to other groups or thoy may bo joined to each other to form a dloulflde bridge. Cysteine or methionine or other anino acids capable of such may bo in different oxidised or reducod states. Other modifications cay bo implied by content horoln.

Tho synthetic thyrocalci onin active s-atorlals of tho procont invention have a shorter chain length than tho 32 amino the British acid corapovm&o defined in/Patent It has been found that tho shortest chain of aaino acido, ctarting from tho amino end, that has beon proven to ehow thyrocaloltonin activity is of 16 amino acido. The activity of theBO aateriale ie lover than that of thyrocalcitonin itcol .

The invention also inoludeo further synthetic thyrocaloitonino with aaino acid chains of from 17 to 31.

In accordance with another aepoct of the present invention there io provided a process for producing a polypcptido having the above defined amino acid constitutional formula, that ies, a polypeptide containing at leact the first 16 amino acids in the eequcnce ^i e abovo, comprising providing a first peptide fragment containing a plurality of arclno acida and a second peptide fragracnt containing at lcaot one amino acid, tho two fragme G having an aaino acid cequsncc such that when coupled together thoy form a polypeptide containing at least the firat 16 araino acids in the sequence defined above, and coupling said frag?-icnts by blocking an a¾ino terminal of ono fragment and tho correoponding carbosy terainal of tho other fragment. It will bo noted that in general tho wy thesiG docs not start at tho beginning, aaino acid 1, and build up tho molecule by adding acid after acid one at a time. On tho contrary, it has boon found arid "more- eco omic l? to synthcsico poptldo fragir.ente of intoraodiate length fror tho original amino acida and thon couple toge he some of theso peptide fr gments to form tho larger iaoloculoa which, beginning vit ono having a chain of the amino acids nuiaberod 1 to 16 in tho above formula, have boon proven to show thyrocalcitonin activity. Fo exam le, typical eynthesee of 16, 22, 25 and 29 amino acid molecules will be deccribed below in the exae-ples. It should bo undorotood that these are only typical procedures which illustrate tho procsao aspoct of the invention without limiting it to exact number© of amino acids in the peptide f gme ts which are aynthcBieed. T e * is, of couroe, an onoraoue number of pocclbilitiee for modifying tho procesc, and in tho preeent application only typloal processes will be described.

In tho synthoeec tho various steps aro usually mm mmlmmm checked by procedures which may include amino acid analysis and thin layer chromatography. In the latter procedure, there are a number of sorbents for coating supports, such as glass plates, which are used in producing the thin layer chromatograms . In general, in the examples two commercially available thin layer chromatography plates are used. They are sold by Analtech, Inc., of Wilmington, Delaware, under their trademark "Uni-plate." One is a silica gel adsorbent containing silica gel and other constituents, sold by Merck & Co. under their designation "Silica Gel GF" and the other Merck's "Alumina GF" . In each case the sorbent composition is attached with a conventional binder and contains a conventional fluorescent material. It should be understood that the invention is not limited to the use of these two particular commercially sold, thin layer chromatography platesj and they are only illustrations of typical thin layer chromatography plates .

Thin layer chromatography utilizes a number of typical solvents or solvent mixtures, one of which consists of 40 parts chloroform, 20 parts methanol, and 4 parts of water. Another solvent mixture is 20 parts chloroform and 10 parts methanol. In the examples when a particular solvent mixture is designated with its parts numbers, it should be understood that this relates to one of the chloroform base mixtures referred to above. Thus, the first one may be designated in the examples 40:20:4. In addition to the chloroform based solvent mixtures, others are also used in the Examples, such as 40 parts n-butanol, 12 parts water and 4 parts acetic acid. This may sometimes be abbreviated BAW, or, alternatively, 40:12:4. Occasionally another solvent mixture of 19 parts ethyl acetate and 1 part acetic acid has also been used. It should be understood that the examples are not intended to be exhaustive of all possible solvent mixtures but are only intended to represent the use of some typical ones which have been found very effective in the syntheses. The invention is, however, of course, not limited to these particular solvent systems .

Thin layer chromatography is sometimes one dimensional and sometimes two dimensional. In the examples one dimensional chromatograms are used. That is to say, a small amount of the mixture to be chromato-graphed, usually 1 to a small number of micrograms, is placed at an origin, and, after development, the distribution of the various constituents then appears as spots in a line, sometimes with tails extending from the spot, usually in the direction opposite to solvent sys-tem movements . Spot tests in thin layer chromatograms are often viewed under ultraviolet light where the particular compounds have fluorescing chromophores which are readily detectable against the background color of the thin layer plate fluorescor.

As in all extensive protein and peptide syntheses, it is often necessary repeatedly to block one or other functional groups, and this is true in the present case also. No unknown blocking techniques are described, and it is an advantage of the present invention that it can use standard methods . Sometimes a blocking group, such as, for example, a p_-nltrobenzyl ester of a C terminal carboxyl group in an amino acid or peptide, fluoresces . When such a chromatogram is viewed under ultraviolet light, it is necessary to distinguish whether the R values relate to the peptide product or to the starting ester.

Often spots are developed by other color producing agents, such as ninhydrin, which reacts with free amino groups. Another is treatment with chlorine gas, which replaces hydrogens in amide groups, and then developing with o-tolidine-potassium iodide. As is usual, this is referred to in the art as tolidine-chlorine .

Another developing agent that is sometimes used is Ehrlich's reagent, £-dimethylaminobenzaldehyde. This is sometimes used specifically to detect tryptophane in the presence of other amino acids.

Quantitative analysis of the large molecules, such as peptide fragments and the like, sometimes presents a problem, both because of the large size of the molecules and the fact that in many cases the amount of material available is quite small. Sometimes an actual analysis of the carbon, hydrogen, nitrogen, sulfur, etc. found will check quite closely with the calculated percentages of some of the elements but not so closely with others. For example, there may be a good correlation for nitrogen and sulfur but not so close a correlation for carbon and hydrogen. In such a case, it is quite common practice for protein chemists to make a check analysis only for the elements where the first analytical figures are not as close .

The active new compounds of the present invention are useful in the treatment, cure, prevention, amelioration or the like of disorders of calcium metabolism including such as osteoporosis . The active new compounds of the present invention may be administered in various well known ways. For example, injectable formulations may be prepared. In such cases, it is com mon to include carrier proteins which often enhance act ivities . This was done in the animal test referred to below .

Because of the vary large number of examples necessitated by the nature of this invention, a number of abbreviations have been used in addition to the normal abbreviations for the various amino acids . Al-though these abbreviations are quite common in the art, the following glossary sets forth the abbreviations used in the examples: OMe O-Methyl OEt O-Ethyl M.A. Mixed anhydride (Usually standing for the method used in a coupling reaction) HOAc Acetic Acid EtOAc Ethyl acetate ET20 Ether. Specifically, diethyl ether NMM N-Methylmorpholine TLC Thin layer chromatography THF Tetrahydrofuran DMF Dimethylformamide OBzl 0-benzyl SBzl S-benzyl ONB 0-p_-Nitrobenzyl BOC t-Butyloxycarbonyl TFA Trlfluoroacetic acid or Tri- fluoroacetate, as tneT case may be .

HOSu N-Hydroxysuccinimide OSu N-Hydroxysuccinimide ester Z Benzyloxycarbony1 Z2 Dibenzyloxycarbonyl Glx Glutamic acid or Glutamine, as the case may be.

Asx Aspartic acid or Asparagine, as the case may be .

O-t-Bu O-t-Butyl DM Dimethylformamide or N,N- Dimethylformamide DCCD Dicyclohexylcarbodiimide /0SO3H Benzenesulfonic Acid CH30SO3H _-Toiuenesulfonic Acid DMAC Dimethylacetamide or N,N- Dimeth lacetamide i-BuOCCl Isobutyl chloroformate (or iso it butyl chlorocarbonate ) i-BuOCOCl (see) i-BuOCCl R.T. Room temperature All amino acids (except glycine, which is not optically active) used were of the L configuration.

The following paragraphs 1 to set forth general methods and conditions for the examples hereinafter . 1. Mixed anhydride coupling reaction. The procedure consists of ( 1 ) forming a mixed anhydride by t reacting an ct-acylamino acid or an a-acylamino peptide with a lower alkyl chloro ormate in the presence of a tertiary amine base, and then ( 2 ) reacting the resulting mixed anhydride with an amino acid derivative having a free amino group or a peptide derivative having a free amino group, to form a peptide. The procedure is described generally in a review by Albertson, Noel P., in Chapter 4 , "Synthesis of Peptides with Mixed Anhydrides", pages 157-355 Organic Reactions Volume 12, John Wiley and Sons, Inc., New York, London, 1962 . This general technique is applied in Example 1 . 2 . Alkaline hydrolysis, or saponification of an ester, a fundamental method of de-esterification des-cribed by Fieser, Louis F. and Fieser, Mary, Organic Chemistry Third Edition, Reinhold Publishing Corporation, New York, 19 6 * on page 178. This general technique is applied in Example 2 . 3 . Acid cleavage of an amino-blocking t-butyloxycarbonyl group, a conventional technique of peptide chemistry, described by Schrfider, Eberhard and L bke, Klaus, in "I. Amino-Protecting Groups", pages 3-51 of Volume I, Methods of Peptide Synthesis, of The Peptides, Academic Press, New York and London, 1965 .

This general technique is applied in Example 3 . 4. Coupling of an N-hydroxysuccinimide ester of an amino acid derivative or of a peptide derivative with an amino acid derivative or with a peptide derivative, a technique described by Anderson, G.W. et al., Journal of The American Chemical Society, Volume 86, pages 1859-1842 (196 ) 5 Ibidem Volume 85, page 3039; ^ Kisfaludy, L. et al. ACTA Chemica Academiae Scientarium Hungaricae Tome 4 , Fascicles 1-2, pages 33-35, (1965); and L8w, Opere Citato, pages 61-66 (1965). This general technique is applied in Example 4.

. Coupling by use of Woodwards reagent, a technique described generally by Schrdder, Eberhard and Ltibke, Klaus in "III, Formation of the Peptide Bond", pages 113-114 of Volume I, Methods of Peptide Synthesis, of The Peptides, Academic Press, New York and London, 196 . This technique is applied in Example . 6. Cleavage of a p_-nitrobenzyl ester by catalytic hydrogenolysis, a conventional technique in organic and peptide chemistry, described generally by Schr5der, Eberhard and Liibke, Klaus in "II. Carboxyl-Protecting Groups", pages 61-62 of Volume I, Methods of Peptide Synthesis, of The Peptides, Academic Press, New York and London, 196 · This general method is applied in Example 6. 7. Cleavage of a benzyloxycarbonylamino acid or of a benzyloxycarbonylamino peptide by catalytic hydrogenolysis, a conventional technique of peptide chemistry, described by Schrfider, Eberhard and Liibke, Klaus, in "I. Amino-Protecting Groups", pages 22-30 of Volume I, Methods of Peptide Synthesis, of The Peptides, Academic Press, New York and London, 196 · This general technique is applied in Example 7. 8. Coupling by use of dicyclohexylcarbodi-imide, a procedure described by Schr6der, Eberhard and Liibke, Klaus in "III, Formation of the Peptide Bond", pages 108-111, of Volume I, Methods of Peptide Synthesis of The Peptides, Academic Press, New York and London, 1965 * as modified by Zimmerman and Anderson (use of N-hydroxysuccinimide) Jour. Am. Chem. Soc . 8 Example 1 Preparation of BOC · al ·Leu ·O B t-Butyloxycarbonyl- -valyl-leucine p_-Nitrobenzyl Ester BOC -Val. OH + Η·Leu-ONB x <|)S03H BOC -Val .Leu -ONB 21.73 Grams (0.10 mole) of BOC -Val* OH, t-butyloxycarb.onyl-valine, was dissolved in 150 milliliters of dry tetrahydrofuran with magnetic stirring and chilled to -17°C, and, then, 11.0 milliliters (0.10 mole) of N-methylmorpholine was added. After a few minutes 13.40 milliliters (0.10 mole) of isobutylchlorofor-mate was added, resulting in immediate precipitation. The mixture was stirred for 5 minutes, during which the temperature rose to -12 °C. Then a solution of 42.5 grams (0.10 mole) of H.Leu ·ONB x (j)S03H, leucine p_-nitrobenzyl ester benzenesulfonate, in 150 milliliters of dry dimethylacetamide containing 11.0 milliliters of N-methylmorpholine was added. The temperature rose to °C. (since the added solution was at room temperature). The mixture was then stirred in the cooling bath for 15 minutes, during which the temperature fell to 0°C. The cooling bath was removed.

About an hour later, the mixture was filtered to remove N-methylmorpholine hydrochloride. The filtrate was concentrated to about 50 ml. with a vacuum evaporator and a hot water bath. Dilution of the residue with about 150 ml. of water precipitated an oil which solidified on scratching. TLC on silica ( 24 chloroform:1 methanol) showed a major product spot at Rf 0.75 (U.V., chlorine-tolidine) . Recrystallization from warm alcohol solution by adding water yielded 36.3 g. of product (78# yield), m.p. 90-91 °, then 104-105 ° .

Example 2 Preparation of B0C -Val .Leu ·0Η t-Butyloxycarbonyl-valyl- leucine N NaOH HC1 BOC-Val-Leu-ONB B0C .Val -Leu -OH EtOH 18.61 Grams (0.040 mole) of the dipeptide ester, t-butyloxycarbonyl-valyl-leucine p_-nitrobenzyl ester, Β00·Val .LeuONB, was dissolved in 100 milliliters of absolute ethanol, and the solution was filtered. 40 milliliters of N aqueous sodium hydroxide was added to the filtrate . After 1 hour at room temperature the reaction mixture was concentrated under aspirator vacuum with a vacuum evaporator warmed with warm water for 15 minutes. Ethyl acetate was added to the residue, and the resulting solution was concentrated again in the same way. More ethyl acetate was added to the concen- trate, and then about kO milliliters of N hydrochloric acid, making the mixture acid. An aqueous layer separated .

The ethyl acetate layer was mixed with about 20 milliliters of water, and 20 milliliters of saturated aqueous sodium bicarbonate was added. The mixture was shaken, and then allowed to separate . The aqueous layer was removed, and the ethyl acetate was extracted twice more with additional portions of saturated aqueous sod-ium bicarbonate . The ethyl acetate residue was set aside .

The aqueous bicarbonate extracts were combined, and N hydrochloric acid was added to acidity, causing separation of an upper ethyl acetate layer and a lower aqueous layer. The layers were separated, and the aqueous layer was extracted again with ethyl acetate, and the ethyl acetate extracts were combined, and the combined ethyl acetate solution was washed twice with water, the last water wash having a pH of 3. The washed ethyl acetate solution was dried and clarified with anhydrous sodium sulfate, and the dried ethyl acetate solution was placed in an evaporating dish.

After evaporation, the residue was partly crystalline, but completed crystallization on stirring, giving a dry yield of 14.5 grams (theoretical, 13.2 grams ) .

The 1 .5 grams of crude product was purified by extraction and re-extraction between ethyl acetate and saturated aqueous sodium bicarbonate and finally acidified to yield some yellow oil and a colorless solid.

This mixture was suspended in water and refiltered. The solid was removed by filtration, washed with water, and dried, giving 8.93 grams of purified tbutyloxycarbonyl-valyl-leucine melting at 157-158°C. An additional 1.10 grams of product melting at 157-157.5 °C. was recovered from the residues.

Example j? Preparation of TFA x Val .Leu ·OH, Valyl-leucine Trifluoro- acetate BOC-Val-Leu-OH ±22^ ^ TFA x H ·Val .Leu ·OH 6.61 Grams (20 mmoles) of BOC^Val'Leu-OH, t-butyloxycarbonyl-valyl-leucine, was put in a flask, and 10 milliliters of trifluoroacetic acid was added. The mixture dissolved with effervescence in a few minutes . After 1 hour the solution was concentrated using vacuum evaporator, a warm water bath, and at first, an aspirator vacuum, then an oil pump vacuum. The residual heavy oil was diluted with a few milliliters of diethyl ether. The resulting mixture was filtered, and the collected solids washed, first with trifluoroacetic-acid-in-ether solution, then with ether. The filtrate and washes were combined, and the resulting solution was diluted with ether while warming, until incipient cloudiness (the volume at this point being about 150 milliliters). Crys-tallization was induced by scratching. The crystallizing mixture was further diluted with ether to about 200 milliliters, and crystallization then was allowed to continue to substantial completion, after which, the crystalline solid was collected by filtration, washed with ether, and dried in an oven, giving batch A, -33 grams, melting at 99-107 °C.

The filtrate was concentrated to small volume, using a vacuum evaporator, and diluted with ether, giving a gummy solid. More ether was added, and the mix-ture was let stand overnight to form a firm solid. This material was collected by filtration, washed with ether, and dried in an oven, giving batch B, 0.86 gram, melting at 99-107 °C. The total of A and B is 6.19 grams, or 90$ of the theoretical yield of 6.88 grams.

By thin layer chromatography on silica, samples of both A and B gave ninhydrin spots at Rf 0.3 in the system 40 parts chloroform: 20 parts methanol: 4 parts water. The filtrate from B gave ninhydrin spots at Rf values 0.48 and 0.8. Tolidine-chlorine gave faint spots at 0.84 for both A and B.

Example 4 SBzl Preparation of B0C -Cy Is -Val -Leu -OH, jt-Butyloxycarbonyl -S-benzyl-cysteinyl-valyl-leucine SBzl †Bzl BOC-Cys-OSu + TFA x H.Val-Leu-OH —3B0C -Cys -Val -Leu -OH 6.00 Grams (17-5 mmoles) of the dipeptide, TFA*Val -Leu -OH, -valyl-leucine trifluoroacetate, and 1.46 grams (17-4 mmoles) of sodium bicarbonate were dis-solved in 10 milliliters of water with effervescence, and the solution was chilled in an ice-water bath while stirring. A solution of 3 -55 grams (8.7 mmoles) of the SB l ester, BOC-Cys-OSu, t-butyloxycarbonyl-S-benzyl-cysteine N-hydroxysuccinimide ester, in 25 milliliters of tetra-hydrofuran, was then added, resulting in a cloudy solu- tion, temperature 12 °C. The cooling bath was removed, and stirring was continued. After 10 minutes thin layer chromatography on silica using the system 40 parts chloroform: 20 parts methanol: 4 parts water showed a major spot at Rf 0.8 — Ο.98 (ultraviolet, positive; tolidine - chlorine, yellow positive), indicating the presence of substantial amounts of the OSu ester starting material, with a minor ultraviolet only spot at 0.25 — > 0.44 and a minor tolidine-chlorine positive spot only at 0.65 . The starting dipeptide showed as a streak (tolidine-chlorine, positive; ninhydrin, positive) at Rf 0.2 — 0.5 , with a minor spot (tolidine-chlorine, positive; ninhydrin, positive) at Rf 0.84. The reaction mixture showed the dipeptide (TFA x H«Val .Leu-OH) , and a double ultraviolet-positive and yellow tolidine-chlorine positive at Rf 0.8 and 0.9 · At 30 minutes a sample showed more of the slower moving spot at Rf 0.85 , and less of the faster moving spot at Rf 0.95 . At this point the pH of the solution was about 6. One equiv-alent (0.73 gram) of sodium bicarbonate was added at this point, causing clearing and effervescence for about 10 minutes . At 55 minutes very little of the faster moving ( ^. 0.95 ) spot was left, and the presumed product spot (yellow) covered R-f Ο.8-Ο.9. Much of the dipeptide spot (Rf 0.2-0.5 ) was still showing. At 70 minutes, a 10$ excess (0.36 gram) of the OSu ester was added, dissolving in a few minutes. Thin layer chromatography on silica using the solvent system 40 parts chloroform: 20 parts methanol: 4 parts water of a sample removed at 80 minutes showed the ninhydrin positive streat at Rf 0.8- 0-0.98, the Rf 0.9 (ultraviolet, positive; tolidine-chlorine, yellow positive) spot, with a faint spot above. At this point the pH was about 6 .5 , but an accurate reading was questionable. About 0.20 milliliter (about 0.4 mmole) of saturated aqueous sodium bicarbonate was added, apparently without e fect . Thin layer chromatography at 90 minutes showed the ninhydrin positive spot and no OSu ester (a sample of known OSu ester showed a yellow spot at Rf Ο.98) .

At 125 minutes 0.5 milliliters of saturated aqueous sodium bicarbonate (approximately 1 mmole) was added, moving the pH more definitely to 7. Then 0.36 gram of the OSu ester, S-benzyl-t-butyloxycarbonyl-cysteine N-hydroxysuccinimide ester, was added, dissolv-ing in about 5 minutes . Thin layer chromatography at 13 minutes showed the ninhydrin positive spot still SBzl present. A sample of BOC-CysOH, t-butyloxycarbonyl-S-benzyl-cysteine, gave a tolidine-chlorine yellow positive spot by thin layer chromatography on silica, and using the same 40: 20:4 solvent, at Rf 0.9^ where the product spot was presumed to be. The Rf 0.9 spot down in the reaction mixture was stronger. It was decided to work up the reaction mixture .

The reaction mixture was concentrated using a vacuum evaporator to remove the solvent tetrahydrofuran. A gummy precipitate formed, which solidified on being worked with a spatula. This solid was collected by filtration, the filtrate showing a pH of 7 · The solid was washed with water. Thin layer chromatography showed a spot at R- 0.8 (ultraviolet, positive; tolidine-chlorine, positive) and a faint tolidine-chlorine positive spot at Rf 0.2 . The filtrate gave a spot at Rf 0.26 which was positive for ultraviolet, ninhydrin, and tolidine-chlorine, a spot at Rf 0 which was positive for ultra-violet and tolidine-chlorine, and a spot at Rf 0.5 which was positive for ultraviolet and tolldine-chlorine (yellow) .

The solid was suspended in 25 milliliters of water and acidified with concentrated hydrochloric acid, changing the character of the material. The solid was collected by filtration and washed 3 times with water, the pH of the final wash being >. The washed solid was placed in the oven to dry, batch A.

The filtrate from A, on acidification, yielded a small amount of gum, batch B. Both A and B gave a single ultraviolet positive spot at Rf 0.3 with thin layer chromatography. Tolidine-chlorine gave three yellow spots, and also a blue spot at R^. 0.26. Batch B had a faint spot at f 0.3 · Amount of product actually recovered is k .93 grams, which is 90$ of the 5 -^9 grams, melting at 103-107°C. The starting OSu ester, t-butyloxycarbonyl-S-benzyl-cysteine N-hydroxysuccinimide ester can be bought from Fox Chemical Company, 1556 Industrial Street, Los Angeles, California 90021 .

Example 5 Preparation of BOC-Asn«Leu«ONB, t-Butyloxycarbonyl- asparaginyl-leucine £-Ni7robenzyl Ester intermediate +H-Leu.ONB BOC-Asn.Leu.ONB To 580 milliliters of acetonitrile was added 11.6 grams (0.050 mole) of t-butyloxycarbonyl-asparagine. The blocked amino acid may be purchased from Fox Chemical Company, 1556 Industrial Street, Los Angeles, California 90021 . To this solution was added 7.0 milliliters of triethylamine and 12 .65 grams of Woodward's Reagent K, N-ethyl-5-phenylisoxazolium-31 -sulfonate, available from Aldrich Chemical Company, Inc., 2371 North 30 Street, Milwaukee, Wisconsin 53210. The mixture was stirred and allowed to stand at room temperature for about one hour, (clear solution).

At the end of this period, about 0.050 mole of the p_-nitrobenzyl ester of leucine was added to the clear solution and this mixture was allowed to stand at room temperature for about one hour. At the end of this period the solid product which formed on standing was separated by filtration, washed with ether and dried at 50°C. The product was recrystallized from 475 milliliters of isopropanol. Yield, 15 -5 grams, m.p. 195-1 7 .5 °C. decomposes.

Example 6 Preparation of BOC«Asn«Leu«OH, jt-Butyloxycarbonyl- asparaginy1-leucine BOCAsn.Leu.ONB ^ BOC «Asn »Leu .OH 9·32 Grams of the dipeptide ester, t-butyl-oxycarbonyl-asparaginyl-leucine p_-nitrobenzyl ester, B0C«Asn.Leu-0NB, was dissolved in a mixture of 250 milliliters of α,β-dimethoxyethane and 70 milliliters of water. About 1 gram of palladium on carbon catalyst was added and hydrogen gas was bubbled through the mixture for two hours . At the end of this period the catalyst was removed by filtration. The filtrate was concentrated under reduced pressure to a point where water began to be removed. Acidification of the concentrate with phosphoric acid precipitated the product which was then separated and dried overnight. Yield 5.4 grams (74$), m«P« 175-176°C. Thin layer chromatography of the product on a silica phosphor plate using a solvent system composed of n-butanol, acetic acid and water, (40:4:12) and chlorine-tolidine indicator gave one spot at Rf 0.76.

Example 7 Preparation of HC1 x H-Oly .Phe.Gly .Pro.0-t-Bu, Glycl-phenylananylglycyl-proline t-Butyl Ester Bydrochloride Z .Gly .Phe .Gly ·Pro ·0-t-Bu ^ HC1 x H ·Gly .Phe .Gly ·Pro .0-t-Bu 8.07 Grams (0.014 mole) of benzyloxycarbonyl-glycyl-phenylalanylglycyl-proline t-butyl ester was dissolved in 200 milliliters of alcohol. About 14 milli-liters of IN hydrochloric acid was added and followed with the addition of about 2.0 grams of 10# palladium-on-carbon. Hydrogen gas was bubbled into the reaction mixture, and carbon dioxide was promptly evolved. The progress of the reaction was followed with thin layer chromatography on silica using a one-to-one isopropanol-heptane mixture. Rf of product is 0.58 as detected with ninhydrin or tolidine-chlorine . All starting material was used after about 1 hour of reaction time. The catalyst was removed by filtration and solvents were removed by evaporation using a vacuum evaporator and reduced pressure .

Example 8 N02 Preparation of Z -His -Arg.Phe .ONB, Benzyloxycarbonyl-histidyl-N-nitro-arginyl-phenylalanine p_-Nitrobenzyl Ester N02 I DCCD Z-His-Arg-OH + H-Phe-ONB x 0S0aH →■ HOSu Clear solutions of 6.2 grams (.0122 mole) of benzyloxycarbonyl-histidyl-N-nitroarginine and 1.4 grams (.0122 mole) of N-hydroxysuccinimide dissolved in 65 milliliters of dimethylacetamide 5.60 grams (.0122 mole) of p_-nitrobenzyl phenylalaninate benzene sulfonate dis-solved in 15 milliliters of dimethylacetamide were combined. 2.52 Grams (.0122 mole) of dicyclohexylcarbodi-imide was added with stirring at room temperature . An additional 1.26 grams of dicyclohexylcarbodiimide and 2.1 grams N-hydroxysuccinimide was added to the mixture and the reaction allowed to continue for 2 hours. Pre- cipitated urea was separated by filtration. The filtrate was concentrated to about two-thirds volume and poured into 400 milliliters of water. The liquid was decanted through a sintered glass funnel. The gummy residue remaining in the beaker and on the funnel was washed with several portions of ether to remove solvent traces. Trituration with a small amount of chloroform, followed by a large amount of ether yielded a solid product, wt. 9.57 grams.

The crude product was purified by washing with about 250 milliliters of ethyl acetate with heating, decanting and triturating from cold ethyl acetate to yield a semisolid. The liquid was again decanted and triturated with ether to give good solidification, wt . 8.1? grams .

Further purification was achieved by boiling the product with about 200 milliliters of chloroform, decanting and washing with cold chloroform. The product was triturated with ether to yield an easily manageable product, wt. 6.82 grams.

Further purification was achieved using a sil-ica gel column (200 grams, 60-200 mesh) and eluting with a solvent system composed of 5$ methanol in chloroform.

Example 9 N0a Preparation of H -His -Arg.Phe -0ΝΒ, Histidyl-N-Nitro- ar in 1-phenylalanine p-Nitrobenzyl Ester 2HBr x H.His. .ONB To a solution of 1.29 grams (0.0014 mole) of histidyl-N-nitro-arginyl-phenylalanine £-nitrobenzyl ester dihydrobromlde in 5 milliliters of water was added an aqueous solution of about 0.6 gram of sodium carbonate. A gummy precipitate was formed which was tritur-ated with ether. Collected Ο.58 gram (6 # of theoretical) of product. Silver nitrate test (presence of halide) on the product was negative. Thin layer chromatography of the product using the solvent system chloroform, methanol and water (20 :10: 2) shows spot at R~ 0.50 .

Example 10 Preparation of B0C Asn Asn Phe OSu t^-Butyloxycarbonyl-asparaginyl-asparaginyl-phenylalanine N-hydroxysuccin- imide Ester Β00·Asn -Asn.Phe.OH + HOSu — B0C -Asn ·Asn -Phe .OSu k .93 Grams ( 0.0205 mole) of t-butyloxycarbonyl-asparaginyl-asparaginyl-phenylalanine was dissolved in 125 milliliters of dimethylacetamide with vigorous stirring. 2 .51 Grams (0.010 mole) of N-hydroxysuccinimide was added to the mixture which was then cooled to about 0-5°C. 4 .15 Grams ( 0.010 mole) of dicyclohexylcarbodi-imide was added to the cooled solution and this mixture was placed overnight in a refrigerator at about 0°C.

Precipitated urea was separated by filtration and the filtrate was concentrated under reduced pressure to about one-third of the original volume . Newly precipitated urea was removed by filtration and the filtrate then concentrated to an oily, semi-solid residue . The residue was treated with ether to yield a crude crys-talline product. The crude product, purified by boiling with about 80 milliliters of tetrahydrofuran and filtering, was thus obtained in about 8o-90# yield, m.p. 199-200°C. effervescence.

Thin layer chromatography on silica of the product using the solvent system chloroform, methanol and water (20 : 10: 2) shows Rf 0.80 spot.

Example 11 OBzl Preparation of BOCSer.Gly ·Μβΐ·ΝΗ», t-Butyloxycarbonyl- O-benzyl-serylgylcyl-methionTne Amide OBzl BOCSer.Gly.Met.H ill MM + i-BuOCCl OBzl 0 (2 ) BOC .Ser .Gly .Met . OCOi-Bu NH3 0 OBzl BOC . Ser .Gly ·Met ·NH2 1.76 Grams ( 0.005 mole) of the BOC tripeptide was dissolved in 25 milliliters of tetrahydrofuran, and 0.33 milliliter (0.003 mole) of N-methylmorpholine was added. This mixture was cooled to -10°C, and 0.40 milliliters (0.003 mole) of isobutyl chloroformate was added. The resulting mixture was maintained at -10°C. for 4 minutes activation, after which gaseous ammonia was bubbled through the mixture.

The mixture was allowed to come to room temperature, causing a crystalline precipitate to form. The solvent tetrahydrofuran was removed under vacuum using a water aspirator, leaving the solid crude product as a residue. This material was washed successively with water, aqueous sodium bicarbonate solution, and then, again, with water. The washed solid was dried under vacuum over phosphorus pentoxide, giving 1.20 grams of solid, crystalline product.

Thin layer chromatography of the product on silica using the solvent system 40 parts n-butanol: 4 parts glacial acetic acid: 12 parts water showed a single spot at Rf 0.86, very faintly positive to ultraviolet, very strongly positive for methionine, and positive (heavy, yellow) to tolidlne-chlorine .

From the aqueous filtrates a second crop of product crystallized out overnight as long needles, melting at 152-153°C. This material had a correct chemical analysis .

Note; The spot test for detecting divalent sulfur (methionine) is a modification of that described by Stephan R. and Erdman, J., Nature, Number 9 8, August , 1964, page 749.

Example 12 SBzlOBzl OBzl SBzl Preparation of BOC-Cys.Ser.Asn.Leu.Ser.Thr.Cys.Val.Leu.

Oa NOa Ser -Ala-Tyr .Try .Arg .Asn.Leu .Asn.Asn.Phe -His .Arg-Phe ·ΝΗ2, t-Butyloxycarbonyl-S-benzyl-cysteinyl-O-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl- N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histldyl-N-nitro-arginyl-phenylalanine Amide A 30 milligram (0.84 micromole) portion of the B0C-22-member-peptide-0NB was dissolved in 13 milliliters of dimethylformamide . The clear solution was saturated with ammonia gas at room temperature and the reaction was allowed to stand at room temperature for 15 hours. Thin layer chromatography on silica using the system chloro-form, methanol, water (20:10:2) indicated only starting material at Rf 0.68 (streak). The reaction mixture was allowed to stand an additional 9 hours at room temperature . Thin layer chromatography on silica in the chloroform, methanol, water (20:10:2) system and in the system n-butanol, acetic acid, water (20:2:6) showed no change at Rf 0.68. A 1 milliliter portion of ethylene glycol saturated with ammonia was added to the reaction mixture and the solution was allowed to stand for 15 hours. Thin layer chromatography on silica using the system n-butanol, acetic acid, water (20:2:6) gave an R 0.68 (minor) and 0.22 (major). After standing for 23 hours more there was no change in the f (same system) so another 1 milliliter portion of ethylene glycol saturated with ammonia was added and the solution was allowed to stand at room temperature for 42 hours. Thin layer chromatography on silica using the system chloroform, methanol, water (20:10:2) gave f 0.25 (major), 0.44 (trace), Ο.85 (trace). The clear brown solution was filtered and the filtrate was concentrated in vacuo. The oily residue was diluted with n-butanol, water (1:1) and concentrated. This dilution and concentration was repeated once. The residue was concentrated in vacuo . The oily residue was then diluted with 25 milliliters of distilled water, cooled to 5°C and placed in a refrigerator at 5°C. for 48 hours. The resulting solid was collected by filtration to give 11.6 milligrams of tan solid.

Example 13 Preparation of HC1 x H-GlyMet «Gl -Phe.Gly -Pro.OH, Glycyl-methionylglycyl-phenylalanylglycyl-proline Hydrochloride BOC -Gly -Met -Gly -Phe -Gly -Pro -O-t-Bu CH3COOH ^ HC1 x H.Gly.Met-Gly.Phe.Gly.Pro.OH .04 Grams (0.007 mole) of t-butyloxycarbonyl-glycyl-methionylglycyl-phenylalanylglycyl-proline t-butyl ester was dissolved with warming in a mixture of 25 milliliters of acetic acid, and 25 milliliters of acetic acid saturated with hydrochloric add was added. The reaction was run under an atmosphere of nitrogen. Carbon dioxide was evolved. After a period of about 90 minutes the product was precipitated with the addition of ether. The mixture was filtered under an atmosphere of nitrogen and the product stored under nitrogen overnight . Thin layer chromatography of the product on silica using a solvent system composed of n-butanol, acetic acid and water (40:4:12) gave a spot at Rj. 0.37 positive to ninhydrin and tolidine-chlorine .

The following examples are prepared according to the methods described in Examples 1 - 13. Only the reactants and products are listed.

Example 14 Preparation of S-Benzyl-cysteinyl-valyl-leucine Tri- fluoroacetate SBzl SBzl BOC Cys Val Leu OH — —> TPA x H Cys Val Leu OH "HrFA Rf 0.5 Example 15 Preparation of t-Butyloxycarbonyl-threonyl-S-benzyl- eysteinyl-valyl-leucine SBzl BOC-Thr.OSu + TFA x H.Cys .Val .Leu ·0Η SBzl BOC «Thr .Cys .Val ·Leu ·OH Rf 0.8 Example 16 Preparation of Threonyl-S-benzyl-cysteinyl-valyl- leucine Trifluoroaceta e SBzl SBzl BOCThr. Cys.Val .Leu-OH — TFA x H-Thr -Cys -Val -Leu-OH +TPA Rf 0.5 Example 17 Preparation of t-Butyloxycarbonyl-0-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucine OBzl SBzl BOC-Ser-OSu + TFA x H.Thr.Cys -Val .Leu.OH Ά'∑' ^ OBzl SBzl BOC ·Ser · hr · Cys .Val .Leu .OH m.p. 175-178°C.

Example 18 Preparation of O-Benzyl-seryl-t reonyl-S-benzyl-cysteinyl- valyl-leucine Trichloroacetate OBzl SBzl BOC -Ser-Thr. C s.Val.Leu.OH ^> OBzl †Bzl TFA x H.Ser-Thr.Cys.Val-Leu.OH Rf 0.6 Example 19 Preparation of t-Butyloxycarbonyl-S-benzyl-cysteinyl-O-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl-S-benzyl-cystelnyl-valyl-leucine SBzlOBzl OBzl SBzl BOC'Cys.Ser.Asn-Leu-OH + TFA x H-Ser .Thr.Cys .Val-Leu-OH -159 C. to R.T. B0C c # Ser Agn Leu Ser Thr m c _Val t M.A.

Leu . OH Rf 0.9 Example 20 Preparation of Asparaglnyl-leucine Trlfluoroacetate R.T.

BOC.Asn.Leu-OH +TFA—> TFA x H «Asn -Le ·0Η Example 21 Preparation of tButyloxycarbonyl-O-benzyl-seryl-asparaginyl-leucine OBzl I 70 C. to R.T.

BOC.Ser.OSu + TFA x H'Asn.Leu«OH —^ OBzl J BOC «Ser -As -Le ·0Η Rf 0.4 - 0.6 Example 22 Preparation of O-Benzyl-seryl-asparaginyl-leuclne Trlfluoroacetate OBzl OBzl I R.T. J BOC .Ser .Asn -Leu -OH +^FA > TFA x H -Ser -Asn -Leu -OH Rf 0.2 Example 22 Preparation of t-Butyloxycarbonyl-S-benzyl-cysteinyl-O- benzyl-seryl-asparaginyl-leucine SBzl OBzl BOCCys-OSu + TFA x H· Ser. Asn· Leu· OH — 5°C. to R.T. ^ SBzlOBzl BOC . Cys . sir· Asn· Leu· OH M.P. 172 - 17^°C.

Example 2k Preparation of li-Butyloxycarbonyl-S-benzyl-cysteinyl- O-benzyl-seryl-asparaginyl-leucine N-Hydroxysuccinimide Ester SBzlOBzl BOC . Cys . Ser .Asn. Leu. OH "17°C ¾to 7°C' > M.A.

SBzlOBzl BOC · Cy Ts · SeIr .Asn «Leu. OSu Rf 0.9 Example 25 Preparation of t-Butyloxycarbonyl-S-benzyl-cysteinyl- 0-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl- threonyl-S-benzyl-cysteinyl-valyl-leucine SBzlOBzl OBzl SBzl BOC Cys .Ser .Asn »Leu. OSu + TFA x H«Ser «Thr -Cys-Val.Leu.OH SBzlOBzl OBzl SBzl R.T. to 58°C. —> BOC · CyIs . SeIr ·Asn ·Leu · SeIr «Thr · CyIs ·Val NaHCOa Leu-OH M.P. 218 - 228°C.

Example 26 Preparation of Dibenzyloxycarbonyl-histidyl-N-nitro- arginine Methyl Ester N02 N0a Za-His.0H + H-Arg.OMe x° ΐΡ Z2 -His -Arg -OMe M.A.

Example 27 Preparation of Benzyloxycarbonyl-histidyl-N-nitro- arginine 0a NOa Za.Hls.Arg.OMe Z-Hls-Arg-OH NaOH M.P. 161 - 173°C.

Example 28 Preparation of Histidyl-N-nitro-arginyl-phenylalanine p-Nitrobenzyl Ester Dihydrobromide Z -His .A -ONB Rf 0.50 Example 29 Preparation of t-Butyloxycarbonyl-asparaginyl-phenyl- alanine p_-Nitrobenzyl Ester BOCAsn-OH + CH3^S03H x H-Phe-ONB —+ Reagent BOC-Asn-Phe-ONB M.P. 191 - 192°C.

Example 30 Preparation of Asparaginyl-phenylalanine p_-Nitrobenzyl Ester Hydrochloride BOC-Asn-Phe-ONB —", > > HC1 x H-Asn-Phe -ONB +HC1 M.P. 184 - 187°C.

Example 31 Preparation of t-Butyloxycarbonyl-asparaginyl-asparaginyl-phenylalanine p_-Nitrobenzyl Ester HC1 x H-Asn.Phe .ONB + BOC .Asn.OH —S-^ + Reagent K BOC «Asn .Asn.Phe .ONB M.P. 209-213°C. eff.

Example 32 Preparation of t-Butyloxycarbonyl-asparaginyl-asparaginyl-phenylalanine BOC.Asn .Asn.Phe.ONB ^0"^' *° 0°C BOc .Asn-Asn-Phe -OH H2,Pd M.P. 204 - 206°C.

Example 33 Preparation of t-Butyloxycarbonyl-asparaginyl-aspara-ginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenyl-alanine p_-Nitrobenzyl Ester NOa BOC -Asn .Asn-Phe-OSu + H .His -Arg -Phe -ONB NO2 BOC «Asn -Asn ·Phe .His «Arg .Phe ·0ΝΒ Rf 0.26, 0. 6* O.80 and 0.95 Example Preparation of Asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine p_-Nitrobenzyl Ester N02 I BOCAsn.Asn.Phe.His.Arg.Phe.ONB __ΛΚ·^' — TFA N&HCO3 N02 I H.Asn.Asn.Phe.His .Arg.Phe.ONB Example 35 Preparation of t-Butyloxycarbonyl-asparaginyl-leucine g-N*itrobenzyl Ester OSO3H x BOC'Asn.OH + H «Leu ·ΟΝΒ R- τ· + Reagent K ^ BOC'Asn«Leu«ONB M.p. 192 - 195°C.

Example 36 Preparation of Asparaginyl-leucine p_-Nitrobenzyl Ester Trifluoroacetate BOC'Asn.Leu-ONB > TFA x H-Asn-Leu.ONB M.P. 128 - 130°C.

Example 37 Preparation of t-Butyloxycarbonyl-N-nitro-arginyl-asparaginyl-leucine p_-Nitrobenzyl Ester N02 BOC-Arg-OH + TFA x H-Asn-Leu-ONB -20°C. to Example 38 Preparation of t.-Butyloxycarbonyl-N-nitro-arginyl-asparaginyl-leucine N02 N02 BOC-Arg.Asn.Leu.ONB S'P > BOC .Arg.Asn«Leu-OH fa Rf 0.65 Example 39 Preparation of ^-Butyloxycarbonyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine £-Nitrobenzyl Ester N02 N02 BOC'Ar Ig»Asn.Leu«OH + H .Asn.Asn.Phe .His •ArIg.Phe ·Ο Β ·Ο Β Rf 0.7 Example 0 Preparation of N-Nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine £-Nitrobenzyl Ester B ·ONB — " ·τ · —— TFA I NaHUOg H ΝΒ Rf 0.2, 0.32 Example 41 Preparation of t-Butyloxycarbonyl-tryptophanyl-N-nitro-arginyl-asparagTnyl-leucyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine £-Nitrobenzyl Ester 02 02 BOC.Try.OSu + H.Arg.Asn.Leu .Asn.Asn.Phe .His .Arg .Phe .ONB Phe-ONB Rf 0.56 Example 42 Preparation of Tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-pheriylalanyl-hlstidyl-N-nitro-arginyl-phenylalanine p_-Nitrobenzyl Ester N02 N02 BOC -Try .Arg.Asn.Leu.Asn.Asn.Phe .Hie .Arg.Phe .O B N02 N02 R.T I I TFA, NAHCOa^ H.Try .Arg.Asn.Leu.Asn-Asn.Phe .His .Arg .Phe -ONB Rf 0.4 Example kj> Preparation of t-Butyloxycarbonyl-alanyl-tyrosine p_- NTtrobenzyl Ester 12°C to 20°C BOC -Ala-OH + CH»0SO3H x H-Tyr-ONB —z : L BOC ·Ala· yr .ONB Rf 0.47 Example 44 Preparation of Alanyl-tyrosine p_-Nitrobenzyl Ester Hydrochloride BOC-Ala.Tyr .ONB +gC > HC1 x H.Ala.Tyr .ONB R 0.60 Example 5 Preparation of t-Butyloxycarbonyl-seryl-alanyl-tyrosine p_-TTitrobenzyl Ester HC1 x H.Ala-Tyr.ONB + BOCSer.OH —'^0' ?° R>T' M.A.

BOC «Ser .Ala -Tyr .ONB Rf 0.7? - 0.92 Example 46 Preparation of t-Butyloxycarbonyl-seryl-alanyl-tyroslne BOC-Ser-Ala- yr.ONB > BOC ·Ser ·Ala · yr ·OH Rf 0.80 Example 7 Preparation of t^-Butyloxycarbonyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine p_-Nitrobenzyl Ester NOz BOC -Ser .Ala «Tyr ·OH + H «Try «Ar Ig .Asn .Le «Asn .Asn .Phe .His .

Asn.

N02 Leu .Asn .Asn .Phe .His .Ar .Phe .O B Rf 0.60 Example 48 Preparation of t-Butyloxycarbonyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro- arginyl-phenylalanine BOC .Ser .Ala .Tyr -Try Phe · N0a R T ONB — ' * ^> BOC'Ser «Ala«Tyr »Try -ArIg.Asn.Leu.Asn.Asn.Phe Rf 0.22, 0.?, 0.80 Example 9 Preparation of t-Butyloxyearbonyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanyl-O-benzyl-serylglycyl-methionine Amide N02 N02 BOC 'Ser .Al -Tyr .Try .Ar .Asn .Leu .Asn .Asn .Phe .His .Ar .

OBzl Phe.OH + TFA x H .Ser .Gly -Met »NHg BOC.Ser.

N02 N02 OBzl I I I Ala · yr . ry .Arg ·Asn ·Leu ·Asn ·Asn ·Phe .His.Arg .Phe . Ser .Gly Met«NH2 R 0.60 Example 50 Preparation of Seryl-alanyl-tyrosyl-tryptophanyl-N-nitro arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-^henylalanyl-O-benzyl-serylglycyl-methionine Amide Tri luoroacetate N02 N02 I I BOC -Ser «Ala «Tyr -Try .Arg.Asn«Leu.Asn ·Asn -Phe «His -Arg.Phe .

OBzl N02 Ser.Gly.Met .NHa R,Tt > TFA x H.Ser .Ala.Tyr .Try .Arg.Asn.

+TFA Oa OBzl I I Leu «Asn .Asn«Phe .His .Arg.Phe.Ser.Gly .Met · Η¾ Rf 0.1 Example 1 t-Butyloxycarbonyl-S-benzyl-cysteiriyl-O-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl- N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl- henylalanyl-O-benzy -serylglycyl-methionine Amide SBzlOBzl OBzl SBzl BOC'C s.Ser.Asniieu.Ser.Thr.Cys.Val.Leu.OSu + H.Ser-Ala.

Tyr -Try .A -Met NO2 NOa Ser -Ala -Tyr «Try ·Ar Ig .Asn .Le .Asn ·Asn · Phe -His ·Ar I · Phe · OBzl Ser -Gly -Me .NH2 Rf 0.5 Example 52 Preparation of t^Butyloxycarbonyl-O-benzyl-serylglycine Methyl Ester OBzl BOC Rf 0.84 Example 53 Preparation of t-Butyloxycarbonyl-O-benzyl-serylglycine BOC-S -OH Rf O.8O Example 5 Preparation of t-Butyloxycarbonyl-O-benzyl-serylglycyl- methTonine Methyl Ester BOC OBzl I BOC ·Ser .Ol .Met ·OMe Rf 0.74 Example 55 BOC ·S .Met .OH Rf 0.82 Example 56 Preparation of O-Benzyl-serylglyeyl-methionine Amide Trifluoroacetate ly .Met · Η2 Example 57 Preparation of Cysteinyl-seryl-asparaginyl-leucyl-seryl-threonyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-arginyl-phenylalanine Amide SBzlOBzl OBzl SBzl BOC -Cys .Ser .Asn .Leu .Ser «Thr .Cys «Val .Leu ·Ser «Ala «Tyr -Try · N02 -60°C. to R.T.

Arg . Asn.Leu-Asn.Asn.Phe.His.Arg.P e. Ha +HF > H · Cys .Ser ·Asn ·Leu .Ser · hr . Cys .Val .Leu . Ser .Ala . yr .Try .

Arg «Asn -Leu.Asn.Asn.Phe .His .Arg.Phe ·ΝΗ2 Example 58 Preparation of Cysteinyl-seryl-asparaginyl-leucyl-seryl-threonyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl- istidyl-arginyl-phenylalanyl-serylglycyl-raethionylglycyl-phenylalanylglycyl-proline Amide Hydro luoride SBzlOBzl OBzl SBzl BOC · Cys . Ser .Asn .Le . Ser .Thr . Cys .Val .Leu .Ser .Ala .Tyr .Try †02 †Oa OBzl Arg .Asn .Leu .Asn .Asn .Phe .His .Arg .Phe .Ser .Gly .Met .Oly .Phe Gly.Pro. Hz ~1Q°^' to R-T- > HF X H -Cys -Ser -Asn -Leu .

Ser .Thr .Cys .Val -Leu ·Ser .Ala -Tyr .Try .Arg .Asn .Leu -Asn .Asn Phe -His-Ar .Phe ·Ser ·Gly ·Met ·Gly .Phe .Gly ·Pro . H2 Example 59 Preparation of Cysteinyl-seryl-asparagiriyl-leucyA^seryl- threonyl-cystelnyl-valyl-leucyl-seryl-alanyl-tyi ayl- tryptophanyl-arginyl-asparaginyl-leucyl-asparaglnyl- asparaglnyl-phenylalanyl-histidyl-arglnyl-phenylarglnyl-15 serylglycyl-methlonine Amide Trlhydrofluoride SBzlOBzl SBzl BOC *Cys .Ser .Asn -Leu ·S r -Cys «Val -Leu -Ser -Ala .Tyr .Try ly -Met ·ΝΗ2 -60°C. to R.T.

+HF 3HF x H'Cys «Ser «Asn«Leu'Ser «Thr -Cys · Val ·Leu ·Ser .Ala «Tyr «Try .Arg .Asn .Leu .Asn .Asn·Phe .His .Arg Phe -Ser .Gly -Met. H2 Example 60 Preparation of Benzyloxycarbonylglyeyl-phenylalanyl- lycine t-Butyl Ester Z.Gly-Phe-Gly-OH + H-Pro-O-t-Bu '^*0' ^° 0°c- ■JQ — M.A.

Z .Gly.Phe .Gly ·Pro .0-t-Bu M.P. 77 - 78°C.

Example 61 Preparation of t^Butyloxycarbonylglycyl-methionylglycyl-phenylalanylglycyl-proline t-Butyl Ester BOC -Gly.Met.OH + HC1 x H.Gly- Phe.Gly.Pro.0-t-Bu BOC ·Gly .Met .Gly ·Phe .Gly ♦Pro ·0-t-Bu DCCD + HOSu Rf 0.85 Example 62 Preparation of t-Butyloxycarbonyl-O-benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl-proline OBzl BOC-Ser-OSu + H ·Gly -Met ·Gly ·Phe .Gly · Pro .OH OBzl BOC .Ser .Gly -Met ·Gly ·Phe ·Gl · Pro ·OH Example 63 Preparation of t-Butyloxycarbonyl-O-benzyl-serylglyeyl-methlonylglycyl-phenylalanylglycyl-proline Amide OBzl I -10°C. to R. .

BOC ·Ser .Gly .Met .Gly ·Phe ·Gly ·Pro .OH M.A., NH3 OBzl BOC .Ser .Gly ·Met ·Gly ·Phe .Gly ·Pro .NH2 Rf 0.57 Example 64 Preparation of O-Benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl-proline Amide Hydrochloride OBzl BOC ·Ser ·Gly -Met .Gly ·Phe .Gly ·Pro ·ΝΗ2 HCl^HOAc—^ OBzl HCl x H.Ser.Qly-Met.01y«Phe.Gl ·ΡΓΟ·ΝΗ2 Example 65 Preparation of t-Butyloxycarbonyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-aspara^inyl-phenylalajyl-histidyl-N-nltro-arginyl-phenylalanine p_-Nitrobenzyl Ester OBzl SBzl N02 BOC'Se Ir. hr.CyIs .Val.Leu.OH + H.Ser -Ala.Tyr .Try .ArIg.Asn.

N02 1 R T ^ Asn'Leu.Phe-Hls.Arg.Phe.ONB DCCD + HOSu ^ OBzl SBzl †02 BOC ·Ser · hr .Cys .Val .Leu .Ser .Ala ·Tyr .Try .Arg .Asn.Leu . 02 Asn»Asn.Phe .His .Arg.Phe ·0ΝΒ Rf 0.80 Example 66 Preparation of O-Benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-trypto-phanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanine p_-Nitrobenzyl Ester BOC ·S NO2 OBzl SBzl Asn-Phe -His -Ar Ig.Phe .ONB —R T \> TFA x H-SeIr .Thr -Cy's . 0a Val «Leu .Ser «Ala «Tyr «Tr ·Arg ·Asn -Leu ·Asn ·Asn «Phe «His · Rf 0.8 Example 67 Preparation of jt-Butyloxycarbonyl-S-benzyl-cysteinyl-O-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalajyl-histidyl-N-nltro-arginyl-phenylalanine p_-Nitrobenzyl Ester SBzlOBzl OBzl †Bzl BOC'Cys «Ser .Asn.Leu-OH + H«Ser -Thr «Cys «Val »Leu «Ser -Ala.

Rf 0.6 - 0.8.

Example 68 Preparation of t^Butyloxycarbonyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanyl-O-benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl-proline Amide BOC'Ser.Ala.Tyr .Try .A .

OBzl OH + HCl x H.Ser.Gly.Met-GlyPhe.GlyPro.NHa—DCCD'+ 'HOSU NOa †02 BOC'Ser.Ala.Tyr.Try .Arg ·Asn·Leu ·Asn ·Asn.Phe .His.Arg .Phe · OBzl Ser .Gl .Met .Gly ·Phe .Gly .Pro .NH2 Rf 0.5 Example 69 Preparation of Seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanyl-O-benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl- proline Amide N02 NO2 BOC 'Ser «Ala .Tyr .Try .Arg.Asn »Leu -Asn.Asn «Phe -His «Arg «Phe · OBzl Ser.Gly.Met.Gly-Phe.Gly.Pro.NH2 HC1R TH0Ac > N02 I HC1 x H ♦Ser .Ala -Tyr .Try .Arg.Asn.Leu -Asn.Asn. Phe -His .

NOa OBzl Arg.Phe -Ser «Gly .Met -Gly .Phe .Gly .Pro ·ΝΗ2 Rf 0.5 Example 70 Preparation of t^Butyloxycarbonyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalajiyl-O-benzyl-serylglycyl-methionyl-glycyl-phenylalanylglycyl-proline Amide OBzl SBzl I I BOC'Ser.Thr.Cys.Val.Leu.OSu + HC1 x H-Ser .Ala.Tyr .Try · A ly ·Met · Gly · Phe · OBzl SBzl Gly.Pro.NH2 —R'T* ^> BOC.Ser.Thr. Cys. Val«Leu. Ser .Ala- N02 N0? OBzl Tyr. Try . Arg. Asn. Leu. Asn· Asn. Phe · His · Ar . Phe . Ser· Gly · Met . Gly- Phe - Gly- Pro- NH2 Rf 0.75 Example 71 Preparation of O-Benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl-trypto-phanyl-Nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanyl-O-benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl-prollne Amide Trlfluoroacetate OBzl SBzl N02 BOC -Se Ir -Thr -CyIs .Val 'Leu «Ser .Ala -Tyr .Try .ArIg .Asn.Leu · N02 OBzl Asn «Asn.Phe .His .Ar Ig.Phe ·SeIr ·Gly .Met ·Gly ·Phe ·Gly ·Pro ·ΝΗ2 OBzl SBzl »-T.

+TFA 7 TFA x H.SeIr «Thr -CyTs .VaLLeu-Ser .Ala«Tyr -Try · N0a N02 OBzl Ar Ig «Asn-Leu ·Asn ·Asn ·Phe «His ·ArI ·Phe .SeIr ·Gly «Met .Gly ·Phe Gly -Pro. H2 Example 72 Preparation of t^-Butyloxycarbonyl-S-benzyl-cysteinyl- 0-benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-seryl-alanyl-trysol-tryptophanyl-N-nitro-arginyl-asparaginyl-leucyl-asparaginyl-asparaginyl-phenylalanyl-histidyl-N-nitro-arginyl-phenylalanyl-O-benzyl-serylglycyl-methionylglycyl-phenylalanylglycyl-proline Amide · SBzlOBzl OBzl SBzl BOC-Cy Is .SeIr -Asn.Leu.OSu + TFA x H *SeIr «Thr «CyTs «Val «Leu · N0a N02 Ser «Ala «Tyr .Try -Ar -Asn «Leu ·Asn «Asn ·Ρηβ .His «Arg ·Phe · OBzl Ser -Gly -Met -Gly -Phe -Gly ·ΡΓΟ·ΝΗ2 'Τ' SBzlOBzl OBzl SBzl BOC ·Cy Is ·SeIr ·Asn ·Leu ·SeIr ·Thr ·CyIs ♦Val «Leu ·Ser ·Ala .Tyr · |100a2 NNO0aa OBzl Try «Arg -Asn -Leu .Asn.Asn ·Phe .His «Ar · Phe ·Ser .Gly .Met Gly ·Phe ·Gly · Pro ·ΝΗ2 Example 73 Preparation of t-Butyloxycarbonyl-tyrosyl-tryptophane BOC.Tyr.Tyr·OH Example 74 Preparation of Tyroayl-tryptophane BOC-Tyr.Try.OH + H.Tyr.Try.OH R^. 0.5, 0.4 and 0.82 Example 75 Preparation of t-Butyloxycarbonyl-alanyl-tyrosyl- tryptophane BOC'Ala.OSu + H«Tyr -Try ·0Η — BOCAla.Tyr -Try ·0Η Example 76 Preparation of Alanyl-tyrosinyl-tryptophane Hydrochloride 1 Ω°Γ to R T BOCAla-Tyr.Try.OH HCI HOAc HC1 X H*Ala'Tyr 'Τγ ,oh Rf 0.2 Example 77 Preparation of ^t-Butyloxycarbonyl-O-benzyl-seryl-alanyl- tyro slny 1-try tophane )Bzl BOC.S fer-OSu + HC1 x H-Ala-Tyr .Try -OH R.T.

Example 78 Preparation of 1;-Butyloxycarbonyl-N-nitro-arginyl-asparaginyl-leucine N-Hydroxysucclnlmlde Ester I -13°C. to 55°C. ^ BOC·Arg ·Asn·Leu ·OH M.A. , HOSu > Rf 0.82 Example 79 Preparation of t-Butyloxycarbonyl-N-nitro-arginyl-asparaginyl-leucine Amide N02 BOC -Arg.Asn -Leu-OSu + NH3 °° ' to 10°C. ^ Rf 0.7 Example 80 Preparation of N-nitro-arginyl-asparaginyl-leucine Amide Trifluoroacetate BOC sn -Leu · H2 Rf 0.25 Example 8l Preparation of t-Butyloxycarbonyl-O-benzyl-eeryl-alanyl-tyrosyl-tryptopKanyl-N-nitro-arginyl-asparaginyl-leucine- Amide OBzl N02 BOC«Se Ir .Ala.Tyr -Try ·0Η + TPA x H ·ArI ·Asn «Leu ·NH2 -Leu ·ΝΗ; Rf 0.8 Example 82 Preparation of O-Benzyl-seryl-alanyl-tyrosyl-trypto-phanyl-N-nitro-arginyl-asparaginyl-leucine Amide Tri luoroacetate BOC TFA x H sn-Leu.NHa Rf 0.7 Example 83 Preparation of t>-Butyloxycarbonyl-0-benzyl-seryl-threonyl-S-benzyl-cysteinyl-valyl-leucyl-O-benzyl-seryl-alanyl-tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucine Amide BOC NO 2 AJrg*AAsn.TLeu«N*mH2 -15-—°CM. Ato R.T. ^? BOC s ·Leu ·NH2 Rf 0.8 Example 84 Preparation of O-Benzyl-seryl-threonyl-S-benzyl- cysteinyl-valyl-leucyl-O-benzyl-seryl-alanyl-tyrosyl- tryptop anyl-N-nitro-arginyl-asparaginyl-leucine Amide OBzl SBzl OBzl N02 i l l 1 BOC -Ser .Thr .Cys .Val .Leu .Ser .Ala .Tyr .Try .Arg .Asn .Leu . H2 R>T< OIBzl SIBzl OIBzl TFA + aHC03 ^ H«Ser «Thr .Cys ·Val.Leu- Ser .Ala. yr .Tr · It . 0.8 Example 85 Preparation of t-Butyloxycarbonyl-S-benzyl-cysteinyl-O- benzyl-seryl-asparaginyl-leucyl-O-benzyl-seryl-threonyl- S-benzyl-cysteinyl-valyl-leucyl-O-benzyl-seryl-alanyl- tyrosyl-tryptophanyl-N-nitro-arginyl-asparaginyl-leucine Amide SBzlOBzl OBzl SBzl OBzl BOC · Cy Is -SeIr ·Asn-Leu*OSu + H«SeIr.Thr.CyIs«Val.Leu«SeIr.Ala.

NO2 SBzlOBzl Tyr .Try .Arg.Asn.Leu ·ΝΗ2 BOC ♦ Cys ·Ser -Asn.Leu.

OBzl SBzl OBzl N02 I I I I Ser ·Thr .Cys .Val ·Leu ·Ser ·Ala·Tyr ·Try ·Ar ·Asn·Leu ·NH2 Rf 0.85 Example 86 Preparation of Cysteinyl-seryl-asparaginyl-leucyl-seryl- threonyl-cysteinyl-valyl-leucyl-seryl-alanyl-tyrosyl- tryptophanyl-arginyl-asparaginyl-leucine Amide Hydro- fluoride 02 Arg.Asn«Leu·ΝΗa "6° to R,T' > HF x H·Cys ·Ser .Asn.Leu Ser ·Thr .Cys .Val .Leu .Ser ·Ala ·Tyr · ry ·Ar ·Asn·Leu ·NH2 Example 87 Redetermination of Thyrocalcitonin Activity of the Pro- duct of Example 86 A sample of the product of Example 86 was dis-solved in 0.01 N hydrochloric acid, and the resulting solution was diluted with water to a concentration of about 0.4 milligram of sample per milliliter of solution (pH about 3) · This solution was tested for the presence of sulfhydryl by a modification of the method of Boyer, P.D., "Spectrophotometry Study of the Reaction of Protein Sulfhydryl Groups with Organic Mercurials," Journal of the American Chemical Society, Volume 76 , pages 3 1-^337 ( 195 ) . The result was positive.

The pH of the solution was then adjusted to about 7 .5 with aqueous ammonia and stirred vigorously causing aeration until the solution tested negative for the presence of sulfhydryl.

The solution was then frozen and lyophilized to a dry powder. This powder was tested for thyrocalci-tonin activity in the usual manner using the rat assay, and the material showed activity at least equal in potency to that of the product of Example 86 .

This powder was subjected to counter current distribution using the solvent system 5 parts n-butanol: 4 parts water: 1 part glacial acetic acid, and at least 3 different products were detected.

Claims (13)

31993/2 WHAT WE CLAIM IS:

1. A polypeptide having the constitutional formula: S S I I . Cys. Ser. Asn. Leu. Ser. Thr. Cys. Val. Leu. Ser. Ala. 1 2 3 4 5 6 7 8 9 10 11 Tyr. Try. Arg. Asn. Leu. (Asn. )„(Asn. ) (Phe. ) (His. ) 1 m n o 12 13 14 15 16 17 18 19 20 (Arg. ) (Phe. ) (Ser. ) (Gly. ) (Met. ).(Gly. ) (Phe. ) p q r " s t u v 21 22 23 24 25 26 27 (Gly. ) w (Pro. ) x 28 29 wherein the N tertrinal group of said polypeptide is a group completing an amino group or a functional derivative thereof, the C terminal of said polypeptide is a group completing a carboxyl group or a functional derivative thereof and each -S. represents a m ere apt o blocking group or when taken together represent an -S-S- linkage and wherein the subscripts 1 to w are zero or 1 with the proviso that when a subscript is zero, the or each subsequent subscript in the amino acid sequence is also zero, whereby the amino acid sequence of the polypeptide is preserved, the dots conventionally representing bonds connecting amino acids or attached to groups thereon.

2. A polypeptide according to claim 1, wherein the C terminal of the polypeptide is an amide and the N terminal is amino.

3. A polypeptide according to claim 1 or claim 2, wherein the subscripts 1 to q are 1 and subscripts r to w are zero or 1 with the proviso that when one of subscripts r to w is zero, the or each subsequent subscript in the amino acid sequence is also zero whereby the amino acid sequence of the polypeptide is preserved.

4. 31992/8

5. in the first step and coupling it with a peptide fragment having a blocked carboxy terminal group and an unblocked amino terminal group and, if necessary, repeating the procedure until a polypeptide having at leasiffche first

6. 16 amino acids in the sequence of the formula of claim 1 is obtained, and recovering the product.

7. A process according to any one of claims 4 to 6 wherein the procedure is repeated until a polypeptide having at least the first 22 amino acids in the sequence of the formula of claim 1 is obtained.

8. A process according to claim 7 wherein the procedure is repeated until a polypeptide having at least the first 25 amino acids in the sequence of the formula of claim 1 is obtained.

9. A process according to claim 8 wherein the procedure is repeated until a polypeptide having at least the first 29 amino acids in the sequence of the formula of claim 1 is obtained.

10. A process according to any one of claims 4 to 9 wherein one fragment having the sequence of amino acids 1 to 4 of the formula of claim 1, is coupled to another fragment having the sequence of amino acids 5 to 16 of the formula of claim 1,

11. A process according to any one of claims 7 to 9 wherein one fragment having the sequence of amino acids 1 to 9 of the formula of claim 1 is coupled to another fragment having the sequence of amino acids 10 to 22 of the formula of claim 1.

12. A process according to claim 4 and substantially as hereinbefore described. - - 31393/2 -PS-*-

13. A olypeptide whenever produced by a process 4 12 according to any ono of Claica -9- to -1-7. S. HOROWITZ & CO. AGENTS FOR APPLICAN TS