9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids

It is a privilege to be able to contribute to this volume in which Professor Zervas’ friends, students, and colleagues have joined together to honor him and his remarkable contributions to peptide chemistry. Although I did not know him until after his days in the Bergmann Laboratory at the Rockefeller Institute, I can claim the distinction of now working in those very same rooms that they occupied back in the mid-1930s. Like all peptide chemists, I am greatly indebted to Professor Zervas, having depended so heavily on the carbobenzoxy group and on the various modified urethan protecting groups which have been direct extensions of the revolutionary advance that Bergmann and Zervas made.

Solid-phase peptide synthesis.

The Isolation of Peptides by High-Performance Liquid Chromatography Using Predicted Elution Positions

C-terminal amidated peptides: production by the in vitro enzymatic amidation of glycine-extended peptides and the importance of the amide to bioactivity.

The structure and mechanism of formation of human calcitonin fibrils.

A detailed account is given of the total synthesis of human calcitonin M, the hypocalcaemic hormone possessing the dotriacontapeptide sequence I. In the build up of I, the protected sequences 1–10 and 11–32 served as intermediates for the preparation of the protected precursor dotriacontapeptide 1–32 (II). While the synthesis of the fragment 1–10 has been reported previously [1], the present account includes a detailed description of the build up of the intermediate 11–32. Racemisation encountered in the synthesis of this fragment is discussed and conditions indicated in which it is minimized.



From the final coupling step using dicyclohexylcarbodiimide-N-hydroxysuccinimide [3], the protected dotriacontapeptide II was obtained pure in a yield of 65% after counter-current distribution.



In the removal by acidolysis of the protecting groups from II even under optimal conditions two side reactions occur: alkylation of the 8-methionine side chain and N to O acyl migration at the serine or threonine residues. By-products from these reactions were removed by counter-current distribution giving very pure I free of diastereoisomers and possessing the biological activity of highly purified natural calcitonin M. In the routine assay [4] after repeated testing its activity was evaluated at 100 ± 5 U/mg (calculated on content of free peptide).

Human calcitonin. VI. Synthesis of calcitonin M

Assays of 8 synthetic analogues of human calcitonin in rats showed that their hypocalcaemic activity was drastically reduced by deletion of the C-terminal amide group, chain-shortening or opening of the disulphide ring, but unaffected or enhanced by modification of the N-terminal amino group.

Struktur-Wirkungsbeziehungen beim menschlichen Calcitonin. III. Die biologische Aktivität verkürzter oder an den Kettenenden modifizierter, synthetischer analoger

Ample evidence has been accumulated to support the assumption that calcitonin preparations obtained from ultimobranchial bodies are generally 20 to 40 times as potent in their hypocalcaemic activity in the rat than those of thyroidal origin. Of the ultimobranchial peptides, only salmon calcitonin (SCT1) has been synthesized so far. The amino-acid sequence of eel calcitonin has been elucidated, and many other avian, reptilian and piscine calcitonins have been extracted and assayed in more or less purified form. All have been found to be very active. Of the four known thyroidal peptides, three displayed low potency of comparable magnitude; as regards the fourth — ovine calcitonin — no activity has been reported.

Analogues of human calcitonin

Relative activities of a series of corticotrophin analogues have been measured by means of five different bioassays using the rat. Similarities in the relative potencies of various ACTH analogues determined using lipolysis or steroidogenesis in vivo and for the lipolytic and steroidogenic responses of fat pads and adrenal slices in vitro emerged and support the concept of a close structural relationship between the ACTH receptors in adipose and adrenal tissues in the rat. Potencies based on the steroidogenic response of isolated adrenal cells, adrenal slices or in-vivo experiments differed markedly from each other. Inactivation of peptides did not occur in the isolated cell assay, so it is likely that this assay estimates potency at the receptor level. A number of arguments suggest that the difference between the isolated cell assay and the other steroidogenic assays lies solely in the effects of peptide inactivation in the latter, and this allows the relative metabolic stabilities for the peptide analogues in these assays to be calculated. In this way it can be shown that: (1) Replacement of L-Ser by D-Ser in amino acid position 1 markedly increases the metabolic stability of the peptide and has only a slight effect on receptor properties. (2) Shortening at the NH2-terminus reduces the activity of peptides at the receptor level by several orders of magnitude, but increases their relative metabolic stability. (3) Introduction of amide groups at the CO2H-terminus markedly increases receptor potency of (1-16), (1-17) and (1-18) ACTH without affecting their metabolic stability in vivo. However, amidation of the CO2H-terminus does have a large effect on metabolic stability in the adrenal slice assay. (4) Replacement of Arg by Lys in positions 17 and 18 of (1-18) ACTH increases potency at the receptor level (adrenal cells) but has little effect on metabolic stability. The comparison of potencies obtained in the various assays, therefore, throws light on the significance of each assay. In addition, the effects of structural modification of analogues can be separately evaluated with respect to the metabolic stability of a peptide and its potency at the receptor level.

Differences between in-vitro and in-vivo potencies of corticotrophins: an interpretation in terms of metabolic stability.

The new hypocalcaemically active peptides of formula I h-cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-P he-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-val-Gly-Ala-Pro-OH and corresponding compounds in which one or more of the asparagine and glutamic acid radicals are replaced by the aspartic acid or glutamic acid radical and/or the aspartic acid radical is replaced by the asparagine radical, their dimers, especially those in which 2 identical peptide sequences (1-32 and 1'-32') are joined in an anti-parallel arrangement via the cysteine radicals 1,7' and 7,1' by means of a disulfide bond, and derivatives are useful as hypocalcaemic agents and are prepared by splitting off groups protecting at least one amino or one carboxyl group or oxidizing the corresponding sulfides to the disulfides or condensing together adequate peptides.

Max Brugger

Papers

Human calcitonin. VI. Synthesis of calcitonin M

Struktur-Wirkungsbeziehungen beim menschlichen Calcitonin. III. Die biologische Aktivität verkürzter oder an den Kettenenden modifizierter, synthetischer analoger

Analogues of human calcitonin

Differences between in-vitro and in-vivo potencies of corticotrophins: an interpretation in terms of metabolic stability.

N-acyl and desamino human calcitonin and analogs thereof