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The last step in the semisynthesis of horse cytochrome c analogues (formation of the bond 65-66) requires the conformation of the complex between two complementary fragments, (1-65) lactone and (66-104). The fragments can be obtained from a limited degradation with cyanogen bromide. The amino component in this reaction can also be obtained from organo chemical synthesis in which the C-terminal fragment (81-104) is required in a selectively protected form. The latter is available from a cyanogen bromide degradation of ubiquitously protected cytochrome c. The details of the protection/deprotection reaction and the properties of nonadecamethylsulfonylethyloxycarbonyl cytochrome c are described.

The syntheses of some derivatives of horse cytochrome c-(66-79)-tetradecapeptide are presented. The syntheses are so designed that analogues of this phylogenetically well preserved sequence can be obtained also. The compounds were intended as synthons for the semisynthesis of 65-homoserine-cytochrome c, which we described earlier. A requisite for this project was the C-terminal tetracosapeptide fragment of the protein, accessible through degradation of cytochrome c with cyanogen bromide. The five epsilon amino groups in this compound are reversibly protected with the 2-(methylsulfonyl)ethyloxycarbonyl function, which is resistant to acid and causes little impairment of solubility. The condensation of the fragments leading to the native sequence of horse cytochrome c-(66-104)-nonatriacontapeptide is presented also. The syntheses were performed using the solution strategy. Some unexpected ring closing reactions involving tyrosine and tert.-butyl prolylasparaginylcarbazate, are described.

The synthesis of the model compound Aloc-Ala-Ala-Dma-Ala-Ala-OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N-alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys-Glu-Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible "structure-breaking" functions.

Two new ACTH-analogues, an octadecapeptide amide and a tetracosapeptide containing the lower homologue of arginine (norarginine) in position 8, have been synthesized by the generally accepted method. Special attention was paid to the synthesis of the required tetrapeptide representing the 7-10 sequence, which was obtained either by direct introduction of L-nitronorarginine or by amidination of the gamma-amino function in a protected peptide containing alpha, gamma-diaminobutyric acid. Biological activity determination showed that the shortening e arginine side chain in position 8 results in the formation of active ACTH-analogues.

The synthesis of p-isothiocyanatobenzoylhomoserine lactone from DL-homoserine as well as from L-methionine is described. The compound constitutes a good tool in the solid phase Edman degradation of tryptic protein fragments terminated by lysine.

Six sulphated octapeptide amide analogues, representing the C-terminal sequence of pancreozymin have been prepared in addition to the natural sequence. The tryptophyl residue which is essential for biological activity was replaced in this series by structurally, closely related analogues each of which resemble the properties of the original moiety to varying extents. The syntheses were so conducted that the C-terminal tetrapeptide amides (modified tetragastrins) could be secured. The results of the measurement of one of the biological parameters are indicated. The activity was determined to elucidate which of the physicochemical properties of the tryptophyl residue is most essential for exertion of biological activity in the described test.

In order to investigate the special function of tryptophan in peptide hormones, six tryptophan analogues have been synthesized, in which structural resemblance has been grossly retained and potentially essential properties have only partially been varied. The L-enantiomers have been isolated after enzymatic digestion of racemic dipeptide derivatives, and charge-transfer properties of the compounds have been studied.

Two analogues of α-MSH are described, in which the tryptophan residue occuring in position 9 of the natural hormone has been replaced by pentamethyl-phenylalanine and phenylalanine, respectively. The analogues were synthesized via a conventional procedure and the [Phe9]-analogue also by a semi-synthetic approach, which demonstrated thefavourable properties of the applied, new amino-protecting Msc function for this purpose. The widely different electron donor properties of the substituted residues were accompanied by a large difference in melanocyte stimulating activity of the analogues. The [Pmp9]-analogue, having donor properties comparable to those of the natural compound, was four to five times more active than the analogue containing the poorly donating Phe residue. The opposite effect was noted in in vivo lipolysis in rabbits.

Application of the 2-methylsulfonylethyloxycarbonyl group for temporary amino protection enables the synthesis from one precursor of des-Nα1-acetyl-α-MSH, the two mono N-acetylated forms (in positions I and II) and the diacetyl form of this tridecapeptide amide. The free tridecapeptide amide, although structurally unrelated to the normal substrate, was recognized by an enzyme occurring in calf eye-lens tissue. The product of the enzymatic reaction was exclusively α-MSH. Partial sequences derived from the N-terminus were less rapidly acetylated or not at all, depending on their chain length. The enzyme, therefore, appears to direct its activity to free N-terminal α-amino groups of peptides exceeding a certain critical chain length. Acetylation of -amino functions did not occur.

The synthesis of a stable substrate for enzymatic Nα-acetylation is described. To this end an analogue of α-MSH-(1–10)-decapeptide with norleucine instead of methionine at position 4 is prepared. t-Butylation of an intermediate Z-Tyr-OMe leads only to about 75% conversion in a few hours' reaction time, and cannot be carried to completion. The [Nle4]-decapeptide is as good a substrate for enzymatic Nα-acetylation as the original decapeptide containing methionine.

Two major fragments of horse heart cytochrome c involving the sequences (1-38) and (60-104) were found to produce a stable complex. The two fragments were devoid of any cytochrome c activity. The complex exhibited a hardly measurable electron transfer capacity with respect to cytochrome c oxidase and missed the 695 nm absorption band. The introduction of tryptophan in position 59 restored the intrinsic activity of the complex to the level of native cytochrome c. This was concluded from the convergence of the Eady-Hofstee plots which extrapolate to the same Vmax at high substrate concentrations. The absorption spectrum of the complex in the ferriform contained a clear absorption band at 695 nm (84% of that found with native cytochrome c). The investigation proves the indispensability of tryptophan in position 59 for the transfer of an electron to cytochrome c oxidase and supports the conclusions of Parr et al. about the existence of two consecutive processes in the folding of the two fragments (vide infra).

We think it worthwhile to other experimenters to mention the following observations. Recently it was noted that gel chromatographic separations of the five fragments resulting from the degradation of cytochrome c by bromocyanogen became dependent on the batch number of the gel. This dependency concerns only separations of unprotected fragments, using 7% (v/v) formic acid as the eluent. The elution profile of protected compounds, which in turn run in 50% (v/v) formic acid, was unaffected.

A method is presented for the high-yield synthesis of Fmoc amino acids, which precludes racemization and over-reaction due to carboxyl activation. A detailed procedure is given for the preparation of Fmoc-ONSu.

A new and efficient procedure for the preparation of protected cyclized and protected symmetrical dimeric peptide disulfides is described. A thiol is immobilized onto a solid phase through coupling of the thiol function with a resin-linked trityl group. Following conventional peptide assembly using the Fmoc-strategy, detachment is performed by oxidation with iodine in a suitable organic solvent. When N,N-dimethylformamide is used as the solvent, and the peptide chain contains an acetamidomethylthio function, located N-terminally in a N alpha-(9-fluorenylmethyloxycarbonyl), or N alpha-tert-butyloxycarbonyl cysteinyl residue, or occurring in the chain, then the corresponding fully protected cyclic peptide disulfide will be obtained in high yield and purity. In other solvents (e.g. dioxane or chloroform-methanol 1:1, v/v), the iodine-mediated oxidation gave not only the cyclic product, but also substantial amounts of the parallel symmetrical dimeric peptide retaining Cys(Acm) at the two identical N-termini.

The synthesis of the model compound Aloc-Ala-Ala-Dma-Ala-Ala-OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N-alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys-Glu-Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible "structure-breaking" functions.

The Mpc-group has a somewhat better stability than the Fmoc-group, resists catalytic hydrogenolysis, is highly stable in acidic media and its elimination product does not polymerize spontaneously. In a direct comparison of coupling efficiencies obtained in solid phase peptide syntheses using Mpc- or Fmoc-amino acids it is shown that the use of Mpc-amino acids leads to better coupling efficiencies and, consequently, a more homogeneous peptide. An improved synthesis of Mpc-ONSu and of Mpc-amino acid derivatives is presented.

The N-terminal pentacosapeptide of Cowpea Chlorotic Mottle Virus (CCMV) was synthesized as the Nα25-acetyl, Cα25-methylamide. The compound contains six arginyl residues, which were incorporated in the peptide chain protected by protonation. The compound was obtained from an azide and a carbodiimide mediated condensation, unifying three fragments. The identity of the compound was proved by amino acid analysis, 13C-n.m.r. and 1H-n.m.r. spectroscopy. It behaved like the native protein in its tendency to interact with viral RNA (fragments).

Two analogues of alpha-MSH are described, in which the tryptophan residue occuring in position 9 of the natural hormone has been replaced by pentamethylphenylalanine and phenylalanine, respectively. The analogues were synthesized via a conventional procedure and the [Phe9]-analogue also by a semi-synthetic approach, which demonstrated the favourable properties of the applied, new amino-protecting Msc function for this purpose. The widely different electron donor properties of the substituted residues were accompanied by a large difference in melanocyte stimulating activity of the analogues. The [Pmp9]-analogue, having donor properties comparable to those of the natural compound, was four to five times more active than the analogue containing the poorly donating Phe residue. The opposite effect was noted in in vivo lipolysis in rabbits.

In order to investigate the possible role of the Trp residue in ACTH as a change-transfer donor in the activation of ACTH receptors, two ACTH analogues (beta-corticotrophins (I-24) with L-Ser1 and D-Ser1, respectively) containing pentamethylphenylalanine instead of Trp have been synthesized. In these syntheses a new, alkaline-labile, amino-protecting group, the methylsulphonylethyloxycarbonyl group, was employed. The association constants of ACTH (I-24) and (Pmp9)-ACTH (I-24) with the water-soluble acceptor paraquat, were nearly equal.

The synthesis of N-acetyl-DL-pentamethylphenylalanine and its resolution with brucine are described. Assignment of the configuration of both antipodes was performed with O.R. D.-measurements and checked by enzymic hydrolysis.