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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Influence of Non-Natural Amino Acids at Position 3 of[Mpa 1 , D-Tyr(Et) 2 ] or [Mpa 1 , D-1-Nal 2 ] Oxytocin onTheir Pharmacological PropertiesVassiliki Magafa 1 , Nikos L. Assimomytis 2 , George Pairas 1 ,Revekka Exarchakou 1 , George K. Daletos 1 , Lenka Borovičková 3 ,Jiřina Slaninová 3 , and Paul Cordopatis 11 Department of Pharmacy, University of Patras, Patras, GR-26500, Greece; 2 Departmentof Mechanical Engineering, TEI of Patras, Koukouli, Patras, GR-26334, Greece;3 Department of Antimicrobial Peptides, Institute of Organic Chemistry and Biochemistry,Academy of Sciences of Czech Republic, Prague 6, CZ-16610, Czech RepublicIntroductionOxytocin (OT) is a cyclic nonapeptide [cyclo(Cys-Tyr-Ile-Gln-Asn-Cys)-Pro-Leu-Gly-ΝΗ 2 ] hormone synthesized in hypothalamus and released into the general circulation fromthe posterior lobe of the pituitary gland. Its major physiological roles are: a) to induceuterine contractions and b) milk injection. Beside these actions, OT is involved in severalother processes, such as vascular and cardiac regulation and sexual, maternal and socialbehavior [1,2]. The role of OT in preterm labor led to the search for and design of syntheticpeptide antagonists as potential tocolytic agents [3]. Moreover, in view of the widespreadOT-related actions, OT antagonists may not only be studied as promising inhibitors ofpreterm labor but may also prove useful in the treatment of dysmenorrhea, benign prostatichyperplasia and psychiatric illnesses such as anxiety, sexual dysfunctions, eating disordersetc. [4]. In this study we synthesized 8 new analogues of [Mpa 1 , D-Tyr(Et) 2 ]OT or [Mpa 1 ,D-1-Nal 2 ]OT containing L-/D-1-naphtylalanine [1-Nal] or L-/D-allylglycine [allylGly] inposition 3. We also studied the effect of modified C-terminal amide on biological potencyof 2 new OT analogues containing L-β-(2-thienyl)-alanine [Thi] and L-allylglycine inposition 3.Results and DiscussionThe new analogues were synthesized by Fmoc solid phase methology [5] utilizing a RinkAmide MBHA and [3-((Ethyl-Fmoc-amino)-methyl)-1-indol-1-yl]acetyl AM resins as solidsupport and diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) as couplingreagents. The cyclization was performed in DMSO/H 2 O (1:4, v/v) for 24-48h [6] oralternatively in AcCN/CCl 4 using tetrabutylammonium fluoride (TBAF) [7]. The analogueswere tested for their potency in two pharmacological assays: a) uterotonic in vitro test inthe absence of Mg 2+ on an isolated strip of rat uterus [8] and b) in the pressor test onphenoxybenzamine treated male rats [9]. In parallel, determination of binding affinity ofthe analogues to cloned human OTR using crude HEK OTR cell membrane preparationwas performed [10]. The biological evaluation of the new analogues is summarized inTable 1. For comparison purposes the activities of the analogues [Mpa 1 , D-Tyr(Et) 2 ]OT (I),[Mpa 1 , D-1-Nal 2 ]OT (II) and [Mpa 1 , D-1-Nal 2 , Thi 3 ]OT (III) [11] are also presented inTable 1. As can be seen from Table 1, replacement of Ile 3 by the L- or D-form of the nonnatural amino acids combined with D-Tyr(Et) 2 or D-1-Nal 2 and Mpa 1 modificationsinfluenced biological activities very differently. Analogues having in position 3 residueL-1-Nal, which has more compact side chain than Ile, exhibited no substantial change inthe antagonistic potency in comparison to analogues I and II having no change in position3. On the other hand, the introduction in position 3 of a residue with less bulky aliphaticside chain than Ile, such as allylGly, had an inconsistent effect; analogue 3 havingcombined substitutions in position 1 (Mpa), in position 2 [D-Tyr(Et)] and in position 3allylGly exhibited slightly improved antagonistic potency in comparison to analogue I,while analogue 7 having combined substitutions in position 1 (Mpa), in position 2 (D-1-Nal) and in position 3 allylGly showed oxytocin antagonistic potency slightly decreased incomparison to analogue II.448