Sarah K. Branch

Synthesis and biological evaluation of α-MSH analogues substituted with alanine

Abstract The influence of single amino acid replacements by alanine on the binding affinity and biological activity of α-MSH in B16 murine melanoma cells has been studied systematically. α-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of α-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4–9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met 4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe 7 , Arg 8 , and Trp 9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

Chiral separations by cyclodextrin-modified capillary electrophoresis: Determination of the enantiomeric excess

The chiral nature of living systems has strong implications for biologically active compounds interacting with them. Consequently, the stereoisomers of drugs can differ in both pharmacodynamic and pharmacokinetic actions. Regulatory authorities encourage the pharmaceutical industry to develop single isomers of chiral drugs. Thus, methods are required which are suitable for stereospecifically evaluating the enantiomers of a drug in quality control of enantiomerically pure drugs, e.g. the determination of the enantiomeric excess (ee). Beside the major methods, chiral HPLC and NMR spectroscopy, cyclodextrin-modified capillary electrophoresis is enjoying increasing popularity in both academic and industrial laboratories. In order to obtain a baseline separation of mixture of a minor and a major isomer which is appropriate for determining the ee, the following parameters have to be optimised: the nature and concentration of the cyclodextrin derivative, the composition of the background electrolyte, the pH value, the organic modifier, and the kind and conditioning of the capillary. In addition, the sensitivity of detection has to be taken into consideration, because a limit of detection of 0.1 percent is necessary to fulfil the requirements of the ICH guidelines. Strategies of method development will be discussed using currently reported examples.

Chiral discrimination of phenethylamines with β-cyclodextrin and heptakis(2,3-di-O-acetyl)β-cyclodextrin by capillary electrophoresis and NMR spectroscopy

The resolution of nine sympathomimetic phenethylamine racemates by beta-cyclodextrin and heptakis(2,3-di-O-acetyl)beta-cyclodextrin has been investigated by capillary electrophoresis and 1H NMR spectroscopy. The NMR and capillary electrophoresis results showed that beta-cyclodextrin probably formed stronger complexes with the amines than did heptakis(2,3-di-O-acetyl)beta-cyclodextrin but was a poorer chiral discrimination agent in both techniques. The addition of heptakis(2,3-di-O-acetyl)beta-cyclodextrin to the capillary electrophoresis buffer gave baseline resolution of enantiomer peaks for seven of the compounds studied while beta-cyclodextrin resolved only three of the racemates.

Effect of β-cyclodextrin acetylation on the resolution of phenethylamines with capillary electrophoresis and nuclear magnetic resonance spectroscopy

The effect of acetylation of β-cyclodextrin (CD) on the chiral discrimination of a series of phenethylamines has been investigated using capillary electrophoresis and nuclear magnetic resonance (NMR) spectroscopy. Therefore, pure, fully derivatized CDs were synthesized. Capillary electrophoresis measurements revealed that the 2,3-diacetylated CD was a better discriminator than the 6-acetylated and the native CD. NMR investigations of the complexes suggest that the phenyl ring of the phenethylamines is inserted in the cavity and the side chain interacts with the hydroxyl rim of the macrocycle. The structures of the complexes indicated by the NMR results were supported by molecular models of the derivatised CDs.

Degradation Pathways of Ampicillin in Alkaline Solutions

Ampicillin trihydrate, sodium salt, in aqueous solution has a pH of about 8. No complete degradation pathway has been proposed to explain the degradation of ampicillin under alkaline conditions and the information available explains the formation of only certain products. The present work was carried out with the aim of providing this information.

Chiral discrimination by NMR spectroscopy of ephedrine and N-methylephedrine induced by β-cyclodextrin, heptakis(2,3-di-O-acetyl)β-cyclodextrin, and heptakis (6-O-acetyl)β-cyclodextrin

NMR spectroxcopy has been used to compare the interaction of ephedrine and N-methylephedrine with β-cyclodextrin, heptakis(2,3-di-O-acetyl)β-cyclodextrin, heptakis(6-O-acetyl)β-cyclodextrin. The stoichiometry of the complexes formed between all three cyclodextrins and N-methylephedrine was found to be 1:1 by UV spectroscopy by means of the Job technique. NMR spectra of the single enantiomers of ephedrine and N-methylephedrine in the presence of all three cyclodextrins gave information about the parts of the ligands which interact differently with the host molecules and may be responsible for the chiral discrimination. To quantify the complex stabilities, binding constants were calculated from the changes in the chemical shifts of the ligand signals upon complexation. Analyses of the coupling constants of both species showed that no significant conformational change occurs upon complexation. ROESY spectra of these optical isomers with all three cyclodextrins provided detailed information about the geometry of the complexes. Different intermolecular cross-peaks between the individual isomers of ephedrine and N-Methylephedrine were found for native β-cyclodextrin and its 2,3-diacetylated derivative but not for 6-acetyl cyclodextrin. Analyses of the intramolecular cross-signals of the ligands confirmed that no significant conformational change occurs upon complexation. Chirality 9:211–219, 1997.

The melanocortin (MC3) receptor from rat hypothalamus: Photoaffinity labelling and binding of alanine-substituted α-MSH analogues

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of α‐MSH, [125I‐Tyr2,Nle4,d‐Phe7,ATB‐Lys11]α‐MSH.SDS‐PAGE followed by autoradiography showed a single band at 53–56 kDA for the native receptor of 35 kDA after deglycosylated with PNGase F, consistent with the predicted cDNA. Receptor binding studies with α‐MSH, γ‐MSH and [Nle4,d‐Phe7]α‐MSH established that α‐MSH and γ‐MSH had similar affinities while [Nle4,d‐Phe7]α‐MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved ‘core sequence’ (‐Met‐Glu/Gly‐His‐Phe‐Arg‐Trp‐) of MSH/ACTH peptides. The binding affinities of alanine‐substituted analogues of α‐MSH were determined to investigate the role of individual residues in ligand—receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

Application of modified cyclodextrins in capillary electrophoresis for enantiomeric resolution of propranolol and analogues

Abstract The enantio-resolution of propranolol and four derivatives was examined by free solution capillary electrophoresis (FSCE) using various cyclodextrin molecules. Of the three modified cyclodextrins investigated, hydroxyethyl-β-cyclodextrin provided the largest chiral resolution values for all five analytes. This may be linked to its extended hydrogen bonding chains on the cyclodextrin rim. Methyl-β-cyclodextrin and heptakis (2,3-di-O-acetyl) β-cyclodextrin gave lower maximum analyte resolutions, probably due to differences in their macrocyclic structure and hydrogen bonding ability. The presence of a bulky, non-polar alkyl grouo on the analytes was found to enhance chiral recognition. Methanol was found to have a varied effect on chiral resolutions, dependent on the type of cyclodextrin and structure of the analyte.

Application of 1H nuclear magnetic resonance spectroscopy to the analysis of β-lactam antibiotics and their common degradation products

The (1)H NMR characteristics of the majority of penicillin and cephalosporin beta-lactam antibiotics in world-wide clinical use are presented. Some of the data are novel and include several high resolution (220, 400 MHz) spectra. The influence of solvent and ionisation state upon spectral parameters is discussed and a scheme of analysis proposed for identifying an unknown beta-lactam sample. Spectral features of common degradation products of benzylpenicillin and other penicillins are provided and the use of (1)H NMR spectroscopy in monitoring the breakdown of penicillin antibiotics described. Other aspects discussed are NMR studies of the stereochemistry, association and protein binding of beta-lactam antibiotics.

Polar organic chiral separation of propranolol and analogues using a ?-cyclodextrin bonded stationary phase

A β-cyclodextrin bonded stationary phase was employed for the enantioresolution of propranolol and several analogues in conjunction with various polar organic mobile phases. The effects of structural alterations in the non-polar regions of the analytes were found to exert profound changes upon chiral resolution and capacity values, indicating that features which cannot hydrogen-bond with the cyclodextrin molecule still play an important role in this chiral recognition process. This was linked to a repulsive steric effect facilitating the necessary conditions for chiral resolution. The degree of ionization of the analytes and the type and concentration of organic modifier used were also seen to influence the analytes1 enantio-selectivity and capacity values.