Hans Rink

Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin.

Abstract A trialkoxy-diphenyl-methylester and amide linkage for solid-phase peptide synthesis with Fmoc strategy is described. Protected peptide esters can be smoothly cleaved with weak acid, resulting in fragments with intact side-chain protection. Mild acidic cleavage of the corresponding peptide amide resins yields peptide amides.

Central actions of somatostatin

Somatostatin (SRIF) was applied microiontophoretically to neurons in the frontal and parietal neocortex, the hippocampus and the striatum of rats anaesthetized with either urethane or chloral hydrate. Qualitatively identical results were obtained under both anaesthetic conditions. In urethane-treated rats SRIF elicited a dose-dependent increase of the firing rate of 74% of the neurons studied in the frontal cortex and of 46% of the neurons studied in the parietal cortex. All cortical cells identified as pyramidal cells were excited. In the hippocampus SRIF provoked excitatory responses in two thirds of all neurons. Six out of the nine cells identified as pyramidal cells were excited by SRIF. In the striatum 80% of all neurons were excited. Following repeated exposure of central neurons to SRIF, the magnitude of the excitatory response gradually diminished, indicating desensitisation. SRIF in concentrations ranging from 10(-8) to 10(-4) M did not interfere with the binding of (3H)-muscimol to GABA receptor sites. The release of GABA from synapses preloaded with (3H-GABA) was not influenced by SRIF in the concentration range from 10(-6) to 10(-4) M. These results indicated that SRIF does not evoke the excitatory responses through attenuation of GABA-mediated inhibition. In conclusion, the findings support the hypothesis that somatostatin may function as a neurotransmitter in the central nervous system.

A new peptidic somatostatin agonist with high affinity to all five somatostatin receptors.

All commercially available somatostatin analogs for clinical use have a preference for some but not all somatostatin receptor subtypes. We describe here the synthesis and evaluation in binding and cAMP assays with cell lines stably transfected with sst(1)-sst(5) of a new type of nonapeptide somatostatin analog with a reduced-sized and stabilized structure, Tyr(0)-(cyclo-D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe) (KE108). All five somatostatin receptors subtypes have an extremely high affinity for KE108, equivalent to SS-28 at sst(1) and two to four times higher than SS-28 at sst(2), sst(3), sst(4) and sst(5). Moreover, the compound has agonistic properties at all five subtypes, since it is able to inhibit the forskolin-stimulated cAMP production in sst(1)-sst(5) cells. It is stable for several hours in human serum. This analog may therefore represent a considerable improvement over commercially available somatostatin analogs as it will target all somatostatin receptor subtypes, a particular advantage for cancer-related applications, as human cancers can express concomitantly several somatostatin receptor subtypes.

‘Antiflammins’: Two nonapeptide fragments of uteroglobin and lipocortin I have no phospholipase A2 ‐inhibitory and anti‐inflammatory activity

The ‘antiflammin’ nonapeptides P1 and P2 [(1988) Nature 335, 726‐730] were synthesized and tested for inhibition of phospholipase A2 and release of prostaglandin E2, and leukotriene C4 in stimulated cells in vitro, and in vivo for anti‐inflammatory activity in rats with carrageenan‐induced paw oedema. Porcine pancreatic phospholipase A2, was not inhibited at concentrations of 0.5–50 μM. Prostaglandin E2, and leukotriene C4 release by mouse macrophages stimulated with zymosan or ATP was not affected up to a concentration of 10 μm, nor was prostaglandin release by interleukin 1β‐stimulated mesangial cells and angiotensin II‐stimulated smooth muscle cells. Both peptides exhibited no anti‐inflammatory activity in carrageenan‐induced rat paw oedema after topical (250 μg/paw) or systemic administration (1 or 4 s.c.). These results do not support the claim of potent phospholipase A2‐inhibitory and anti‐imflammatory activity of the ‘antiflammins’ P1 and P2 [1].

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin‐specific inhibitor [(1989) J. Biol. Chem. 264, 7141‐7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N‐terminal core domains (r‐Hir1–43 and r‐Hir1–52) derived from recombinant hirudins and synthetic C‐terminal peptides (Hir45–65 and Hir52–65) ‐ all fragments form complexes with thrombin — we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C‐terminal region. Since hirudin C‐terminal peptides were shown to bind to a non‐catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10‐16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.

Conversion of NGurethane protected arginine to ornithine in peptide solid phase synthesis

Abstract It is shown that Di-Adoc or Boc as guanidino protecting groups do not prevent the acylation and the subsequent conversion of arginine to ornithine in Fmoc solid phase peptide synthesis.

A new combination of protecting groups and links for encoded synthetic libraries suited for consecutive tests on the solid phase and in solution

SummaryA strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.

A Large Fragment Approach to Gene Synthesis

Abstract The total synthesis of a 232 base-pair coding sequence of the proteinase inhibitor eglin c from only six synthetic fragments is described.