A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech.

AbstractPreviously reported studies of the iodine oxidation of S‐trityl‐cysteine peptides and S‐acetamidomethyl‐cysteine peptides, leading directly to cystine peptides, have been extended. Detailed investigations have been made of the reactivities of the S‐trityl and the S‐acetamidomethyl group towards iodine in various solvents. In chloroform, methylene chloride, trifluoroethanol, and hexafluoroisopropyl alcohol the differences in the reaction rates of the two groups have been found to be extremely large, allowing the selective conversion of the tritylthio groups to disulfides in the presence of the S‐acetamidomethyl derivatives. In a second group of solvents, consisting of methanol, acetic acid, dioxane, and mixtures of these solvents with water, simultaneous iodine oxidation of S‐trityl‐ and S‐acetamidomethyl‐cysteine peptides leads to a preferential combination of these two residues, resulting in predominantly asymmetrical cystine derivatives. ‐ The suitability of the two sulfur‐protecting groups in the synthesis of cyclic cystine peptides has been assessed. ‐ Possible reaction mechanisms are discussed. ‐ The scope and limitations of iodine oxidation in peptide synthesis have been studied.The applicability of the method has been demonstrated in the preparation of the open‐chain asymmetrical cystine peptide 5, the protected somatostatin derivative 17, and the A(1–13) segment 19 of human insulin, previously employed in the total synthesis of this hormone.

AbstractCystine peptides are conveniently prepared from S‐acetamidomethyl‐ or S‐trityl‐protected cysteine derivatives by direct oxidation with iodine. Since the reaction proceeds through the formation of sulfenyl iodides, these highly reactive groups may substitute the indole ring of tryptophan residues, resulting in the formation of 2‐thioethers. During the synthesis of the peptide hormone somatostatin, we investigated this possible side reaction. By‐products of the tryptophan‐2‐thioether type can be produced under conditions which lead to a marked retardation of the disulfide bond formation. The largest amount of these compounds were formed when the oxidation was carried out in 90% aqueous trifluoroethanol.In model peptides in which tryptophan and cysteine residues were separated by 1 to 4 glycine residues, the ring size of the resulting thioether exerted a strong influence on the yield: in peptides with 1 and 2 glycines, only dimeric disulfides were formed. Incorporation of 3 and 4 glycine residues gave thioethers in yields of about 40% and 70% respectively. Conversely, under normal conditions of iodine oxidation, when disulfides are rapidly formed from the S‐acetamidomethyl‐ or S‐tritylcysteine residues, tryptophan‐2‐thioethers are produced only in insignificant amounts or not at all.

The effects of two [D-Cys14]-analogues of somatostatin on basal plasma levels of glucagon, insulin and glucose were determined in unanaesthetized rats to re-examine a glucagon-selective action of these peptides which has been claimed by others. Somatostatin, [D-Cys14]-somatostatin and [D-Trp8, D-Cys14]-somatostatin caused a short-lasting, dose-dependent decrease of plasma glucagon and insulin but they had no significant influence on plasma glucose. Glucagon and insulin reached the nadir 2 min after intravenous injection of the peptides (dose range 1--10 micrograms/kg) or 5 min after subcutaneous administration (30 and 300 micrograms/kg). At the nadir, insulin was decreased to a greater extent than glucagon and the effecer the nadir and at high doses, the time-course of some effects of the analogues on either glucagon or insulin differed from that of somatostatin. Thus, these [D-Cys14]-analogues may show partial kinetic dissociation of effects on glucagon and insulin but they are not truly selective inhibitors of glucagon release.

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.

Five analogs of human insulin with d -Cys in different positions (A 6 , A 7 , A 11 , A 6+11 , B 7 ) have been synthesized by the fragment condensation approach, combined with selective disulfide formation. All of them have physicochemical properties noticeably different from those of human insulin. They possess very low biological activity (0.03−1.2%, glucose oxidation in rat fat cells). In contrast, the potency for antibody binding ranges from 7 to 70% of that of insulin. The two analogs with d -Cys in positions A 6 and A 7 have been obtained in crystalline form.

Chemischer InformationsdienstVolume 8, Issue 11 Natural Products ChemInform Abstract: SYNTHESIS OF HUMAN INSULIN. III. PREPARATION OF THE PROTECTED, DOUBLE-CHAIN A(14-21) - B(17-30) FRAGMENT B. KAMBER, B. KAMBERSearch for more papers by this authorB. RINIKER, B. RINIKERSearch for more papers by this authorP. SIEBER, P. SIEBERSearch for more papers by this authorW. RITTEL, W. RITTELSearch for more papers by this author B. KAMBER, B. KAMBERSearch for more papers by this authorB. RINIKER, B. RINIKERSearch for more papers by this authorP. SIEBER, P. SIEBERSearch for more papers by this authorW. RITTEL, W. RITTELSearch for more papers by this author First published: March 15, 1977 https://doi.org/10.1002/chin.197711373Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume8, Issue11March 15, 1977 RelatedInformation

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.

AbstractDie letzten Stufen der Synthese vonHumaninsulin (VI) bestehen in der Kombination der bereits synthetisierten Komponenten (II) (sog.

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.