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Title: Synthesis and evaluation of novel enkephalin analogues.
Author: Smith, Helen.
Awarding Body: University of Wolverhampton
Current Institution: University of Wolverhampton
Date of Award: 1998
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Continuous flow, solid (gel) phase peptide synthesis was adapted in the successful preparation of leucine enkephalin, DEL T I and two novel analogues of the deltorphin peptide, (L-Ala)2 DELT I and (Aza-Ala)2 DELT 1. Peptides were synthesised by sequential N-terminal deprotection and coupling steps. The coupling step of the deltorphin analogue synthesis incorporating the second valine amino acid residue, was modified to minimise the possibility of diketopiperazine (DKP) formation. The pentapeptide was synthesised on a resin support and the dipeptide, Z-Tyr(BzI)-Aza-Ala, was coupled via an isocyanate intermediate, to produce the novel peptide (Aza-Ala)2 DELT 1. Structural confirmation of the peptides was demonstrated by 13C-NMR studies and amino acid analysis of the (Aza-Ala)2 DELT I analogue. Ovine aminopeptidase activity was measured using a colorimetric assay which produced p-nitroaniline. Sensitivity of the assay was increased five to six fold by derivatisation of the p-nitroaniline to an azo dye. Purification of a soluble aminopeptidase from ovine cerebra was developed from Hersh and McKelvy's 1981 bovine preparation. The ovine enzyme was found to possess significantly different properties to those of bovine cerebra. Ovine aminopeptidase has a molecular weight of circa 220 kDa, with molecular weight of SDS PAGE subunits being, 43.5, 49, 69, and 83 kDa, in contrast with the single polypeptide bovine cerebral aminopeptidase of 100 kDa. The ovine aminopeptidase is unstable and loses activity on storage. Its activity could however, be retained for a longer period of time by storage in 10% v/v DMSO. Leucine enkephalin and the DEL T analogues were tested in vitro for biological activity by monitoring the inhibition of electrically stimulated contractions in Guinea Pigileum. Commercial leucine enkephalin was affected most in the presence of naloxone (a J..l-opioid receptor antagonist) giving a Ke value of 1.850 nM. The analogues of deltorphin were all slightly affected by the presence of naloxone, suggesting they might be acting via other opioid receptor populations present in the ileum. The deltorphin analogue Ke values were 2l.223 nM [DELT I], 4.231 nM [(L-Ala)2 DELT I] and 2.519 nM[ (Aza-Ala)2 DELT I]. Pilot studies to investigate the affinity of these peptides to the <5-opioid receptor were performed courtesy of Dr. I. Kitchen (University of Surrey). The DEL T I behaved as a <5-opioid receptor ligand, whilst (L-Ala)2 DEL T I showed little effect at these receptors. The (Aza-AlaY DEL T I showed a high affinity for the <5 receptor. Furthermore displacement of naltindole by (Aza-Ala)2 DEL T I may suggest a subdivision of the <52-opioid receptor further, an effect not previously observed
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry Biochemistry