Peptides: Proceedings of the Sixth European Symposium, Athens, September 1963

Peptides covers the proceedings of the Sixth European Peptide Symposium, held in Athens, Greece on September 1963. This symposium brings together numerous works on the synthesis, reactions, biological activity, and physico-chemical properties of peptides. This book is organized into seven sections encompassing 46 chapters. The first three sections describe the methods of peptide synthesis, racemization, and degradation of peptide chains. These sections examine the developments in non-enzymatic selective modification and cleavage of peptides, as well as the oxidative modification of specific peptide chain. The succeeding section highlights the total synthesis of natural peptides and peptide analogues and the evaluation of the structure-activity relationships and biological properties of these peptides. These topics are followed by discussions on the synthetic pathways and properties of certain special peptides, along with the accompanying synthesis problems with uncommon amino acids and abnormal peptides. This final section explores the gas-chromatographic studies on the physic-chemical properties of peptides. This book will prove useful to organic chemists, biochemists, and peptide researchers.

• Language: English
• Publisher: Elsevier Science
• Release date: Jun 28, 2014
• ISBN: 9781483223544

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SECTION I

METHODS OF SYNTHESIS

Outline

Chapter 1: SYNTHESIS OF ARGINYL PEPTIDES THROUGH ORNITHYL PEPTIDES

Chapter 2: USE OF THE S-ETHYLCARBAMOYL GROUP FOR PROTECTION OF THE THIOL FUNCTION OF CYSTEINE

Chapter 3: FISSION OF TOSYLAMIDE GROUPS WITH METALS IN LIQUID AMMONIA

Chapter 4: REDUCTION OF NITROARGININE DERIVATIVES: INTERMEDIATES AND SIDE REACTIONS

Chapter 5: ÜBER DEN SCHUTZ DER HYDROXYLGRUPPE DES SERINS MIT DEM p-CHLORBENZYL-RADIKAL

Chapter 6: CHROMOGENIC ACID-LABILE PROTECTING GROUPS FOR USE IN PEPTIDE SYNTHESIS

Chapter 7: SYNTHESE VON POLYPEPTIDEN OHNE ISOLIERUNG DER ZWISCHENPRODUKTE

Chapter 8: KERNSUBSTITUIERTE CARBOBENZOXY-SCHUTZGRUPPEN. VERGLEICHENDE UNTERSUCHUNG DER ACIDOLYTISCHEN SPALTUNG

Chapter 9: CYCLISIERUNG VON PEPTIDEN, BESONDERS AN THIOÄTHERN

Chapter 10: DIE AKTIVIERUNG VON ACYLAMINOSÄUREN UND ACYLPEPTIDEN MIT DICYCLOHEXYLCARBODIIMID

Chapter 11: ZUR SYNTHESE VON HYDROXYAMINOSÄUREPEPTIDEN

Chapter 12: CATALYSIS OF PEPTIDE SYNTHESIS. FACILITATION OF ESTER AMINOLYSIS BY BIFUNCTIONAL CATALYSTS VIA A CONCERTED DISPLACEMENT

Chapter 13: PEPTIDSYNTHESEN MIT HILFE VON PROTEOLYTISCHEN ENZYMEN (PLASTEINREAKTION)

Chapter 14: SYNTHESIS AND SOME PROPERTIES OF DISERYLPYROPHOSPHATES

Chapter 15: THE PARTICIPATION OF THE AMIDE GROUP IN THE SOLVOLYSIS OF PHOSPHORIC ACID TRIESTERS DERIVED FROM SERINE AND ETHANOLAMINE

Chapter 16: NEW METHODS IN PEPTIDE SYNTHESIS

Chapter 17: DISCUSSION ON METHODS OF SYNTHESIS

SYNTHESIS OF ARGINYL PEPTIDES THROUGH ORNITHYL PEPTIDES

M. BODANSZKY, C.A. BIRKHIMER, S. LANDE, M.A. ONDETTI, J.T. SHEEHAN and N.J. WILLIAMS,     The Squibb Institute for Medical Research, New Brunswick, N. J.

Publisher Summary

This chapter discusses the synthesis of arginyl peptides through ornithyl peptides. One of the problems in the application of derivatives of ornithine in peptide synthesis is the extreme ease with which most of them cyclize to a lactam. The active ester p-nitrophenyl α-benzyloxycarbonyl-δ-phthalyl-L-ornithinate (I) is prepared by phthalylation of the copper complex of L-ornithine with Nefkens’ reagent followed by carbobenzyloxylation and finally conversion to the p-nitrophenyl ester. Three biologically active arginine-containing peptides can be selected as models for the synthetic approach, namely, arginine vasopressin, arginine vasotocin, and the shortest sequence of α-MSH, or of ACTH, which shows melanocyte stimulating activity, namely, histidyl-phenyl-alanyl-arginyl-tryptophyl-glycine. In the synthesis of the pituitary hormones, the active ester I is allowed to react with glycinamide, the resulting protected dipeptide is treated with HBr in acetic acid. For the removal of the phthalyl group, hydrazine can be added to the solution of the protected nonapeptides in dimethylformamide, while a mixture of methanol and chloroform can be used as a solvent in the case of the protected pentapeptide.

THE difficulties in the synthesis of arginine-containing peptides are well known [1] and although there are several methods which solve this problem, even the one most frequently applied, the use of nitroarginine derivatives [2–7], does not always give satisfactory results. For instance, in the synthesis of bradykinin and some of its analogues [8, 9] the acylation of the imino group of proline by benzyloxycarbonyl nitro-L-arginine proceeded so poorly (Table 1) that a renewed effort toward the synthesis of arginyl peptides seemed to be justified. As early as 1949, J. Fruton pointed out: Another possible approach to the synthesis of arginyl peptides, which remains to be explored, may be the prior synthesis of the corresponding ornithyl peptides … followed by the conversion of the δ-amino group to a guanido group by treatment with cyanamide, guanidine, S-methylisothiourea or O-methylisourea. [10] This approach has been applied in a few instances, but not in a way which could be extended to general use.*

TABLE 1

Acylation of Proline by Z-Nitroarginine

One of the problems in the application of derivatives of ornithine in peptide synthesis is the extreme ease with which most of them cyclize to a lactam [11, 12]. It seemed to be desirable, therefore, to choose for the protection of the δ-amino group of ornithine a masking group which not only can be selectively removed at the appropriate stage of the synthesis, but also gives full protection against unwanted acylation, including cyclization. The phthalyl group suggested itself for this purpose and the active ester p-nitrophenyl α-benzyloxycarbonyl-δ-phthalyl-L-ornithinate (I) was prepared by phthalylation of the copper complex of L-ornithine with Nefkens’ reagent [13] followed by carbobenzyloxylation and finally conversion to the p-nitrophenyl ester [14].

Three biologically active arginine-containing peptides were selected as models for the synthetic approach just outlined: arginine vasopressin [15], arginine vasotocin [16] and the shortest sequence of α-MSH, or of ACTH, which still shows melanocyte stimulating activity, namely histidyl-phenylalanyl-arginyl-tryptophyl-glycine.

In the synthesis of the pituitary hormones, the active ester I was allowed to react with glycinamide, the resulting protected dipeptide was treated with HBr in acetic acid and the synthesis continued in a manner similar to that described earlier in connection with the synthesis of oxytocin [17]. The nitrophenyl ester method [18] was used in all the coupling steps. A reaction time of about 2—4 hr was found to be sufficient for completion of the acylation reactions at room temperature and all the protected intermediates were obtained in high yield, analytically pure, and, with two exceptions, in crystalline form. Properties of the protected peptide intermediates of the synthesis of the pituitary hormones are summarized in Tables 2 and 3; those of the synthesis of the pentapeptide with MSH activity in Table 4.

TABLE 2

Protected Intermediates of Arginine Vasopressin and 8-L-Ornithine Vasopressin

TABLE 3

Protected Intermediates of Arginine Vasotocin and 8-L-Ornithine Vasotocin

TABLE 4

Protected Intermediates of Histidyl-phenylalanyl-ornithyl-tryptophylglycine

For the removal of the phthalyl group, hydrazine (3 moles) was added to the solution of the protected nonapeptides in dimethylformamide, while a mixture of methanol and chloroform was used as a solvent in the case of the protected pentapeptide. This reaction seems to go to completion in 3 hr at room temperature. The guanylation reaction was carried out with the aid of 1-guanyl-3, 5-dimethylpyrazole [19] in dimethylformamide. Removal of the remaining protecting group followed conventional routes: sodium in liquid ammonia in the case of the pituitary hormones, hydrobromic acid in acetic acid in the case of the pentapeptide. In the latter it was found to be of advantage to remove the t-butyl ester group from the C-terminal of the peptide prior to guanylation.

Treatment of samples of the dephthalylated nonapeptides with sodium in liquid ammonia gave the hormone analogues 8-L-ornithine vasopressin and 8-L-ornithine vasotocin (8-L-ornithine oxytocin). Both analogs* exhibit considerable biological activity (cf. Table 5). While the pentapeptide was purified and secured in homogeneous form, the two pituitary hormones and their respective ornithine analogues were so far obtained in crude form only; therefore, their activities as reported in Table 5 will need to be corrected after the isolation of the four cyclic octapeptides in pure form.

TABLE 5

Biological Activities in the Rat Pressor Test (of the Crude Products after Removal of the Protecting Groups)

The authors express their gratitude to Dr. B. Rubin and Miss H. Waugh for the determination of activities in the rat pressor assay and to Mr. J. Alleino and his group for the microanalyses herein reported.

References

1. GREENSTEIN, J.P., WINITZ, M. Chemistry of Amino Acids. New York: J. Wiley, 1961; 1068.

2. BERGMANN, M., ZERVAS, L., RINKE, H. Z. Physiol. Chem, 1934;224(40)

3. HOFMANN, K., RHEINER, A., PECKHAM, W.D. J. Am. Chem. Soc, 1953;75(6083)

4. HOFMANN, K., PECKHAM, W.D., RHEINER, A. J. Am. Chem. Soc, 1956;78(238)

5. VAN ORDEN, H., SMITH, E. J. Biol. Chem, 1954;208(751)

6. ZAHN, H., DIEHL, J.F. Angew. Chem, 1957;69(135) ZAHN, H., DIEHL, J.F. Z. Naturforsch, 1957;12b(85)

7. IZUMIYA, N., MAKISUMI, S. J. Chem. Soc. Japan, 1957;78(1768)

8. BODANSZKY, M., ONDETTI, M.A., SHEEHAN, J.T., LANDE, S. Ann. N.Y. Acad. Sci, 1963;104(24)

9. BODANSZKY, M., SHEEHAN, J.T., ONDETTI, M.A., LANDE, S. J. Am. Chem. Soc, 1963;85(991)

10. FRUTON, J.S. Advan. Protein Chem, 1949;5(64)

11. RUDINGER, J. Collection Czech. Chem. Commun, 1959;24(95)

12. BODANSZKY, M., BIRKHIMER, C.A. Chimia (Aarau), 1960;14(368)

13. NEFKENS, G.H.L. Rec. Trav. Chim, 1960;79(688)

14. Biochem. Preparations, 1962;9(110)

15. DU VIGNEAUD, V., LAWDER, H.C., POPENOE, E.A. J. Am. Chem. Soc, 1953;75(4880)

16. KATSOYANNIS, P.G., DU VIGNEAUD, V. J. Biol. Chem, 1958;233(1352)

17. BODANSZKY, M., DU VIGNEAUD, V. Nature, 1959;81(5688) BODANSZKY, M., DU VIGNEAUD, V. J. Am. Chem. Soc, 1959;81(5688)

18. BODANSZKY, M. Nature, 1955;175(685)

19. HABEEB, A.F.S.A. Can. J. Biochem. Physiol, 1960;38(493)

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*In the conversion of poly-DL-ornithine to poly-DL-arginine by E. Katchalski and P. Spitnik (Nature 164, 1092 (1949); J. Am. Chem. Soc. 73, 3992 (1951)) and in the transformation of tyrocidin to the corresponding arginine analog by H. N. Christensen (J. Biol.Chem. 160, 75 (1945)) no advantage was taken of the selectively removable protecting groups, whilein the guanylation of Nα-tosylornithylglycine by B. C. Barrass and D. J. Elmore (J. Chem. Soc. 1957, 3134), the choice of protecting groups does not allow execution of this conversion at an appropriate stage of the synthesis of a longer peptide chain.

** During the preparation of this manuscript, it was learned that R. L. Huguenin and R. A. Boissonnas (Helv. Chim. Acta 46, 1669 (1963)) reported the preparation of the same two hormone analogues

USE OF THE S-ETHYLCARBAMOYL GROUP FOR PROTECTION OF THE THIOL FUNCTION OF CYSTEINE*

ST. GUTTMANN,     Sandoz Ltd., Basle, Switzerland

Publisher Summary

This chapter discusses the use of the S-ethyl carbamoyl group for protection of the thiol function of cysteine. A large number of biologically important peptides such as ribonuclease, insulin, oxytocin, and vasopressin contain one or several cystine residues in their sequences. During the synthesis of such peptides, the thiol group of cysteine is blocked because of its high reactivity and oxidizability. If several cysteine residues are present, it can be necessary to deblock them not simultaneously but gradually one after the other. For this purpose, selectively removable thiol blocking groups are needed. The ethylcarbamoyl group, which is very easily introduced by treating dry cysteine hydrochloride with ethyl isocyanate, can be a useful protecting group for the thiol function of cysteine. The synthesis of glutathione is an example of simultaneous hydrolytic cleavage or S-carbamoyl and ester groups. The N-Z-group can be cleaved before or after the elimination of the S-carbamoyl and ester groups.

A LARGE number of biologically important peptides, such as ribonuclease, insulin, oxytocin, vasopressin, etc., contain one or several cystine residues in their sequences. During the synthesis of such peptides the thiol group of cysteine has to be blocked because of its high reactivity and oxidizability. In addition, if several cysteine residues are present it may be necessary to deblock them not simultaneously, but gradually one after the other. For this purpose selectively removable thiol blocking groups are needed.

At the present time the universal protecting group for the thiol function is the benzyl group [1]. Its introduction is easy and it is stable under most conditions used in peptide synthesis. Unfortunately its removal requires very drastic treatment with sodium in liquid ammonia. This also splits most protecting groups used for the other functional groups of the molecule. In addition, fragmentation of the molecule may occur if bonds such as Lys-Pro or Tyr-Pro are present [2].

During recent years some other groups have been proposed for the protection of the thiol group: p-nitrobenzyl [3], tetrahydropyranyl [4], benzylthiomethyl [5] and 2, 2-dimethylthiazolidine [6]. Their selective removal under relatively mild conditions is theoretically possible, but no or very few examples of their use in peptide chemistry have been reported.

Very recently the diphenylmethyl [7], triphenylmethyl [7–9], t-butyl [10], benzyloxycarbonyl [11, 12], acetyl [12] and benzoyl [12] groups have been proposed for the protection of the thiol group of cysteine. According to the authors [7, 12] a judiciously combined use of them should allow their partial or simultaneous removal.

In the present paper we wish to report the results of attempts in our laboratories to find improved protecting groups for the thiol group. We looked for a group which would be stable under the acidic conditions necessary for the removal of most N-protecting groups and which would nevertheless be removable under very mild alkaline conditions.

One of the most interesting groups for this purpose could be the S-Z-group proposed by Katchalsky et al. [11]. Its introduction and removal are quite easy, but its sensitivity to hydrogen bromide in organic solvents does not allow selective removal of the N-Z-group in all cases. In the first series of experiments we replaced the highly sensitive benzyl group by other less sensitive alkyl groups.

S-Ethoxycarbonyl-, S-octyloxycarbonyl-, S-cyclohexyloxycarbonyl-, and S-phenylethyloxycarbonyl-L-cysteine were synthesized. Certain difficulties were encountered in their N-carbobenzoxylation and the isolation of pure N-Z-S-alkoxycarbonyl-L-cysteine was rather laborious, the yield rarely exceeding 50 per cent. N-Alkoxycarbonyl-L-cysteine and cystine, formed by an S→N transposition could be identified in the mother liquors. Even when we succeeded in obtaining pure N-Z-S-alkoxycarbonyl-L-cysteine and coupled it to an amino-acid derivative or to a peptide, partial or total S→N transposition occurred when we deblocked its amino end to build up the peptide chain by stepwise addition to it of other amino-acid residues. It was very difficult to isolate the desired peptides in good yields (Fig. 1).

FIG. 1

It is known that because of serine’s hydroxyland amino groups’ favourable steric disposition, its O-acyl derivatives undergo a rapid O→N transposition as soon as the amino group is not protonated [13]. The same transposition will occur with the S-acyl [14] and S-alkoxycarbonyl-cysteine derivatives. S-Acyl-cysteine is an activated thioester and this type of ester is known to undergo ammonolysis still more readily than the O-esters.

In order to make this transposition more difficult, one should attach to the carbonyl group a substituent which could hamper attack on it by the neighbouring amino group. The alkyl substituted carbamoyl group should fullfil this requirement, for in the carbamoyl group the carbonyl function is stabilized by resonance and attack by the neighbouring amino group becomes rather difficult (Fig. 2).

FIG. 2

We found that the ethylcarbamoyl group, which is very easily introduced by treating dry cysteine hydrochloride with ethyl isocyanate [15], could be a useful protecting group for the thiol function of this amino-acid. The crystalline S-ethylcarbamoyl-L-cysteine was converted in nearly quantitative yield to its N-Z derivative (Fig. 3). This latter can be converted to an activated ester or directly coupled using mixed anhydride or dicyclohexylcarbodi-imide with an amino-acid derivative or a peptide. The peptide obtained can be decarbobenzoxylated and coupled in the presence of a tertiary base with an N-protected amino-acid or peptide by the usual methods (e.g. mixed anhydride, dicyclohexylcarbodi-imide or activated ester).

FIG. 3 Synthesis of N-Carbobenzoxy-S-ethylcarbamoyl-L-cysteine

If the S-ethylcarbamoyl cysteine is not in an N-terminal position, even peptides with a free amino group may be prepared without danger of transposition (see Fig. 6 and Table 2). The behaviour of the S-ethylcarbamoyl group is indicated in Table 1.

TABLE 1

Behaviour of S-Ethylcarbamoyl Group under Conditions used in Peptide Chemistry

TABLE 2

Characteristics of Peptides obtained during the Synthesis of Oxytocin

*Isolated as the hydrobromide.

FIG. 6

It can be seen that the group is stable under acidic conditions. This enables the selective cleavage of a large number of N-protecting groups (e.g. Z-, BOC-, trityl, etc.) and that of some other S-protecting groups (e.g. trityl, Z, etc.). It is easily split under basic conditions, e.g. ammonolysis, hydrazinolysis, alkali catalyzed alcoholysis and saponification. For higher peptides longer splitting time is required. The rate of splitting can easily be followed by titration with iodine or