A Comparative Study on Selectivity of α-Conotoxins GI and ImI Using Their Synthetic Analogues and Derivatives

Comparative structure-function studies have been carried out for -conotoxin GI acting on nicotinic acetylcholine receptors (AChR) from mammalian muscles and from the electric organ of the Torpedo californica ray and for -conotoxin ImI, which targets the neuronal a7 AChR. A series of analogs has been prepared for this purpose: chemically modified derivatives, including a covalently linked dimer of GI, as well as analogs wherein one or several amino acid residues have been changed using solid-phase peptide synthesis. The activity of all compounds was assessed in competition with radioiodinated and/or tritiated -conotoxin GI for binding to the membrane-bound AChR of Torpedo californica. Binding of radioiodinated -conotoxin GI dimer was also monitored directly, revealing the largest, as compared to all other analogues, difference in the affinity between the two binding sites in the receptor (K_D 11 and 1200 nM). Comparison of binding data with the results of CD measurements point to important role of the spatial organization of the -conotoxin second loop in manifestation of their muscle or neuronal specificity.     

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.     

Photoactivatable Analogues of α-Conotoxins GI and MI and Their Interaction with Nicotinic Acetylcholine Receptor

Two photoactivatable analogues of -conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, -conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N of Gly1 or N of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native -conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [¹²⁵I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the -conotoxin molecule.     

New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for (2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-abelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl (2-6)-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported KM values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors. © 1998 Rapid Science Ltd     

Identification and quantification of brassinolide-related steroids in the insect gall and healthy tissues of the chestnut plant

Castasterone and 6-deoxocastasterone were identified by GC/MS and/or selected ion monitoring not only in the insect gall of chestnut but also in the healthy tissues, including the shoot, leaf and flower bud. In addition, the gall was found to contain a small amount of brassinolide. Brassinone, which had been reported to be present in the gall, could not be detected in these tissues. The contents of castasterone, 6-deoxocastasterone and brassinolide, which were determined by using selected ion monitoring, are discussed.     

Modern and ancient olive stands near Sagalassos (south-west Turkey) and reconstruction of the ancient agricultural landscape in two valleys

Aim To study the present‐day olive stands and their ecology in the eastern part of the territory of the ancient city of Sagalassos, to study the variation of olive pollen production and dispersal near the olive stands, to establish a modern pollen reference model, and to compare Hellenistic–Roman pollen data from two wetlands with this modern reference model. Location Eastern part of the territory of the ancient city of Sagalassos, western Taurus mountain range in south‐west Turkey. Methods The study is based on field survey, pollen analysis of surface samples, multivariate statistics of modern pollen data and the use of ‘modern analogues’ in comparison with Hellenistic–Roman pollen samples. Results A field survey revealed the presence of 35 olive stands in the study area. These are mainly small‐scale stands. The olive pollen representation in the surface samples is highly variable. Two groups of modern ‘olive’ pollen spectra could be distinguished: (1) a group representing mainly olive stands from lush and moist mixed orchards; and (2) a group representing mainly olive stands from open small‐scale olive stands in combination with annual crop agriculture. Although no ‘perfect’ modern analogue was found for the Hellenistic–Roman pollen data, the fossil pollen data show similarities with modern spectra from the second group, due to the presence of relatively high pollen values for secondary anthropogenic indicators. Main conclusion A well‐organized and diverse, but time‐ and energy‐consuming, agricultural system was maintained nearby the wetlands of Çanakl? soils, presumably to maximize the yields in both valleys.     

Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH(6-13): comparison of two i-->i + 4 lactam cyclization procedures.

The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N-terminus of human growth hormone (hGH), namely hGH[6-13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse beta-turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric byproducts associated with the preparation of cyclic hGH[6-14] peptide analogues based on an i-->(i + 4)Lys-->Glu or Glu-->Lys cyclization strategy.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹ = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.     

辅因子S-腺苷-L-甲硫氨酸甲基类似物及其应用

甲基化在生物学过程中发挥着重要作用。S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine, SAM)作为一种广泛存在于生命体中的辅因子,是大多数生物甲基化反应的甲基供体。SAM依赖型甲基转移酶(methyltransferases, MTase)通过将甲基从SAM分子特异性转移到底物,从而改变底物分子的各种理化性质和生物活性。近年来,许多具有替代甲基取代基的SAM类似物被合成并应用于甲基转移酶,以将不同修饰的基团特异性地转移到甲基转移酶的底物上,从而引入标记官能团或者新的烷基修饰。本文主要综述了近年来该领域不同SAM甲基类似物在合成和应用方面取得的进展,并对这一领域未来的研究方向进行展望。