SummaryPreviously 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(l-13) segment 19 of human insulin, previously employed in the total synthesis of this hormone.Some years ago, we reported that S-trityl-cysteine peptides and S-acetamidomethyl-cysteine peptides [2] could be directly converted into cystine peptides by oxidation with iodine [3] [4]. In the meantime, this method has been applied by us and by others to the synthesis of a variety of structurally different cystine peptides PI ~61. I)Abbreviations are according to the IUPAC-IUB Commission on Biochemical Nomenclature [I]. In addition, the following abbreviations have been adopted in the text: Acm = acetamidomethyl; HFIP= hexafluoroisopropyl alcohol; TFE= trifluoroethanol; DMF = dimethylformamide.
Purpose: Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity.We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. Experimental Design: Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1-to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. Results: High affinity to all receptor subtypes was found. Y III -KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout.The kidney uptake was high but blockable by coinjection of lysine.Conclusion: This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as Somatostatin [somatotropin release-inhibiting factor (SRIF)]is a tetradecapeptide with potent inhibitory actions on several tissues, such as the pituitary, the endocrine pancreas, and the gastrointestinal tract. SRIF also acts as neurotransmitter and neuromodulator (1, 2). The inhibition of the many secretory processes of hormones, such as growth factors, insulin, and glucagon, is an important property of SRIF and is mediated by at least five SRIF receptor subtypes (sst1-sst5), which belong to the G protein -coupled receptor family. Because of the short plasma half-life of <3 min, does not have therapeutic potential in the treatment of conditions related to pathologic secretory processes. Therefore, metabolically stabilized somatostatin-based peptides were developed (3 -7). Two of them, octreotide (Sandostatin) and lanreotide (Somatuline), are being used in the clinic. They display high affinity only to sst2 and moderate affinity to sst3 and sst5. It is therefore conceivable to search for somatostatin analogues with a higher affinity binding profile to all five receptor subtypes (pansomatostatin character) with the aim to improve the established therapeutic potential or discover new indications. Recently, several such molecules have been discovered and developed, such as the cyclohexapep...
The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin"-65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-PhePro-Arg-Pr~-(Gly)~-hirudin~~-~' (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (Pl-Pl') seems to be cleaved.
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