Bernhard Riniker

Protection of carboxamide functions by the trityl residue. Application to peptide synthesis

Abstract Carboxamide functions may be tritylated by an acid-catalyzed reaction with triphenylmethanol and acetic anhydride in glacial acetic acid. The ω-trityl group of asparagine and glutamine is cleavable by TFA, but stable to strong mineral acids in aqueous solution, as well as to nucleophiles and bases. In peptide syntheses, it is ideally suited for combination with side-chain protections of the t.butyl-type.

A General Strategy for the Synthesis of Large Peptides: r1~he Combined Solid-Phase and Solution Approach.

Abstract In the synthesis of large peptides. the yield and purity of the end-products will be greatly improved when smaller segments are purified prior to their use for fragment coupling either on a solid-phase resin or in solution. The Naα-Fmoc(9-fluorenylmethoxycarbonyl) method allows the selective acidolytic cleavage of fully protected peptides with a free α-carboxyl group from the solid-phase resin. For this cleavage, the highly acid-labile HMPB linker, 4-(4-hydroxymethyl-3- methoxyphenoxy)-butyric acid, has been developed. The lipophilic protecting groups, in particular Trt on asparagine, glutamine, and histidine, as well as Pmc (2,2,5,7,8-pentamethylchroman-6- sulfonyl) on arginine, confer a good solubility on most protected peptide segments in organic solution and enable their purification by silicagel chromatography. Whereas the addition of segments on solid-phase resins is often difficult, they can as a rule be coupled easily in solution to give products in high yield and purity. The combined solid-phase and solution strategy is illustrated by the syntheses of human calcitonin-(l-33), human neuropeptide Y, and the sequence 230-249 of mitogen-activated 70K S6 kinase. Large peptides are built up in solution from smaller segments with lipophilic protecting groups, which can be prepared by solid-phase synthesis. The scheme below depicts the synthesis of human NPY using this method.

Protection of histidine in peptide synthesis: A Reassessment of the trityl group

Abstract The trityl (Trt) group is ideally suited for the side-chain protection of His in peptide syntheses, in combination with 9-fluorenylmethyloxycarbonyl (Fmoc) in N α - and protecting groups cleavable by mild acidolysis in other positions of the peptide. 2,4,5-trichlorophenyl (Tcp)- and pentafluorophenyl (Pfp)-esters of Fmoc-His(Trt)-OH and Trt-His(Trt)-OH are strongly activated, but stable compounds. N α -Trt is selectively removable in the presence of N Im -Trt.

[Structure-activity relationship of human calcitonin. III. Biological activity of synthetic analogues with shortened or terminally modified peptide chains (author's transl)].

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.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.

Treatment of adjuvant arthritis in rats: Vaccination potential of a synthetic nonapeptide from the 65 kDa heat shock protein of mycobacteria

Adjuvant arthritis induced by mycobacteria in rats is a widely used model of chronic arthritis. A previously described nonapeptide (Thr-Phe-Gly-Leu-Gln-Leu-Glu-Leu-Thr, amino acid sequence 180-188) from the recombinant 65 kDa heat shock protein of Mycobacterium bovis BCG, which was found to contain a T-cell epitope recognized by both arthritogenic and protective T-cell clones in vitro, has been investigated for its vaccination and therapeutic potential in adjuvant arthritis in rats. The nonapeptide was found not to be arthritogenic, although the T cells from nonapeptide immunized rats cross-react in vitro with mycobacterial antigens. Intraperitoneal administration of 0.1 mg nonapeptide in oil at day -20 or days -2, -1 and 0, resulted in a marked reduction of incidence and severity of adjuvant arthritis. The disease process and severity were also influenced by therapeutic treatment with 0.1 mg nonapeptide injected intraperitoneally at days 7 to 10. Interestingly, subplantar or intravenous application of the nonapeptide had no influence on the disease process. Deletion of the N-terminal threonine led to complete loss of in vivo activity of the nonapeptide.

Identity of structure of ovine and bovine ACTH: correction of revised structure of the ovine hormone.

The amino acid sequences of the pituitary hormone corticotropin (ACTH) from four different mammalian species (porcine [l] , ovine [2,3], bovine [4,5], and human [6] ) have been elucidated, some of them almost twenty years ago. The proposed structures, however, contained a few errors which remained undetected until the amino acid sequences of porcine and human ACTH were recently corrected by two different groups [7,8]. As a consequence of these studies C. H. Li was prompted to re-examine his earlier proposals for the ovine and bovine hormones [9]. In both cases, Edman degradation of the tryptic fragments 2239 showed that the amino acid sequence 25-32 was not correct and therefore had to be revised. According to these new findings, the hormones of the two species should differ in position 25, with aspartic acid in ovine and asparagine in bovine ACTH. Glutamine has been proven to be present in both species in position 33 [3,51. It is known from the early work on sheep ACTH that this compound is unstable under alkaline conditions and that it easily undergoes deamidation [lo]. The same applies to porcine and human ACTH, since both contain the very labile A&-GlyZ6 sequence. The revised structure postulated by Li [9] for sheep ACTH, however, could not explain this lability. It seemed therefore most probable to us that ACTH from this species should also contain asparagine in position 25 instead of aspartic acid and thus should be identical with beef ACTH. The deamidation of asparagine peptides in alkaline conditions leads, via the succinimide derivatives as intermediates, to a mixture of (Yand /I-aspartic peptides, in which the latter predominate. The difficulties

Analogues of human calcitonin I. Influence of modifications in amino acid positions 29 and 31 on hypocalcaemic activity in the rat

The principal sites of calcitonin biosynthesis are the C-cells of the thyroid gland in mammals and the ultimobranchial body in birds, reptiles and fish. The amino acid sequences of the thyrogenic calcitonins from four species are known and two, the human [l] and the porcine [2] hormones, have been synthesized [3,4,5]. Attempts to elucidate the sequence of and synthesize ultimobranchial peptides, on the other hand, have up to now only succeeded in the case of salmon calcitonin [6,7], though others have been isolated in highly purified form. The calcitonins so far obtained from ultimobranchial glands exhibit greater biological activity in rat assays than dhose of thyroid origin [8]. This must be attributable to some particular amino acids, since the sequential differences between the thyroid hormones, which are all roughly equal in activity, are as great or even greater than the differences between them and the ultimobranchial peptides.