Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids.

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.

Synthesis of somatostatin and [D-Trp8]-somatostatin.

Experimental details for practical syntheses of somatostatin and D-Trp8-somatostatin are described. The peptides were assembled from three fragments which permit further syntheses of analogs with modifications at positions 1, 2 or 8. N alpha-Bpoc protecting groups were used for the two major fragments and these were selectively removed in the presence of the tert.-butyl derived amino acid side chain functionalities. The two cysteine residues were protected by acetamidomethyl groups. All the peptide intermediates were fully characterized and a 10-g synthesis of the protected tetradecapeptide is reported. Major fragments were coupled by the azide method in good yield. Dihydrosomatostatin and D-Trp8-dihydrosomatostatin were isolated, purified, characterized and cyclized. Polymeric side-product was successfully recycled (by reduction with dithiothreitol and reoxidation) to give an overall yield for the oxidation of 52%. Somatostatin and D-Trp8-somatostatin were purified by gel filtration or countercurrent distribution and the final products were fully characterized and determined to be > 97% pure by reversed phase high performance liquid chromatography.

Activity and work during pregnancy and the postpartum period: a cross-cultural study of 202 societies.

Cross-cultural information on 202 traditional societies was perused for data on customs surrounding the expected work load of pregnant and postpartum women. The most common single pattern of work activity during pregnancy was that of continuing full duties until the onset of labor. A bare majority of societies did encourage a lightening of work at some time during pregnancy. Postnatally, most societies did restrict maternal work activity; however, few suspended usual work duties for prolonged periods of time. About one half of societies expected a return to full duties within 2 weeks. Results suggest that the current American trend toward increased participation of women of childbearing age in the work force may be in keeping with the work loads of women in traditional societies.

Catalytic hydrogenolysis in liquid ammonia. Cleavage of Nalpha-benzyloxycarbonyl groups from cysteine-containing peptides with tert-butyl side chain protection. Application to a stepwise synthesis of somatostatin.

To exemplify the extension to synthesis of sulfur-containing peptides of the Nalpha-benzyloxycarbonyl and side chain tert-butyl protective group combination, somatostatin has been synthesized via incremental chain elongation starting from the COOH-terminal cysteine. Cleavage of the Nalpha-benzyloxycarbonyl groups was achieved in high yield, at each stage, by palladium-catalyzed hydrogenation in liquid ammonia. All side chain functionalities including the cysteine thiol groups were blocked by tert-butyl-derived groups. Each gave rise to individual n.m.r. signals which permitted sensitive characterization of all intermediate protected peptides. Mild conditions were used for the removal of all protecting groups from the completed somatostatin tetradecapeptide. The tert-butyl thiol protective groups were readily and completely cleaved by mercuric acetate at pH 4. Oxidation of dihydrosomatostatin with potassium ferricyanide provided somatostatin in good yield. The results indicate that the absolute selectivity between alpha-amine and side chain protective group cleavage afforded by Nalpha-benzyloxycarbonyl hydrogenolysis in the presence of omega-tert-butyl groups may now be extended to synthesis of cysteine- and methionine-containing peptides.

Purification of protected syntheic peptides by preparative high performance liquid chromatography on silica gel 60.

A simple preparative system is described for rapid and efficient purification of protected synthetic peptides on a gram scale by high performance liquid chromatography on prepacked silica gel 60 columns. A variety of protected peptides up to tetradecapeptides have been chromatographed at pressures of 50 to 150 psi and obtained in analytically pure from within 2 to 4 h. With such commonly used protecting groups as N-benzyloxycarbonyl (Z), N-2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc), N-t-butyloxycarbonyl (Boc), O- and S-t-butyl (But), and S-acetamidomethyl (Acm), compounds were sufficiently soluble in chloroform, alcohols, acetic acid, or mixtures of these solvents for column loading. Dimethylformamide was also used as a solvent for loading. Solvent systems for column elution in isocratic, stepwise, or gradient modes were composed of chloroform, isopropanol, ethanol, or methanol and acetic acid in ratios that differed for each protected peptide depending on Rf values on t.l.c. plates. A simple chromatography is described which was self-assembled using standard instruments commonly in use in most laboratories. A shut-off valve was designed to prevent loss of material between fractions.